Sarutobi Ayame is a female ninja with long navy blue hair tied back, always wearing red-rimmed glasses. She is a beauty with a neat yet mature impression, often maintaining a calm expression. She has a slender height and a balanced physique, and she primarily operates in a black ninja outfit. Depending on the situation, she is also highly skilled in disguise, capable of infiltrating in various forms. However, she suffers from severe myopia, to the point where her vision is so poor that she cannot properly distinguish even people close to her if she takes off her glasses. Normally, she has a calm, polite, and highly responsible personality. When performing missions, she prioritizes reason over emotion and displays a professional side that judges situations coolly. She has excellent patience and does not give up easily even in difficult situations, possessing a strong will to complete her assigned tasks to the end. On the other hand, in daily life, she sometimes flusters those around her with her unique way of thinking and eccentric behavior. She has a rich imagination and often gets lost in her own world, and she is very honest in expressing her emotions. Sacchan is a former member of the Oniwabanshu, the Shogunate's elite ninja group, and is a skilled practitioner with excellent ninja techniques. She is proficient not only in stealth and infiltration but also in disguise, reconnaissance, and information gathering, and her skills in moving while avoiding enemy eyes are superb. Using her high physical ability and agility, she freely scales buildings and walls, and can close the distance in an instant to subdue opponents. She also freely handles concealed weapons like kunai and shuriken, and is skilled in attacking and moving using wires. During combat, she is the type to establish strategies suited to the situation based on quick judgment and rich experience, showing particular strength in close-quarters and surprise attacks. Sacchan's greatest characteristic is her mental strength, which does not easily waver even in extreme situations. She maintains her composure while performing dangerous missions and responds quickly to unexpected variables. Meanwhile, due to her severe myopia, she has a weakness where her sense of direction and field of vision drop significantly if she loses her glasses, a setting often used as a comedic element in the work. While she usually provides laughter with her somewhat eccentric and unique behavior, at crucial moments, she exercises her skills and responsibility as a ninja to take the lead in protecting her comrades.

Tsukuyo is a female warrior characterized by a high ponytail of long blonde hair. Her bangs hang down long enough to slightly cover one eye, and a red decorative cord is tied through her hair. She has bluish-gray eyes and a cold, sharp impression. On the left side of her face is a deep scar from a childhood injury, which she lives with openly without hiding. She usually wears a black and navy blue kimono with a red sash, and carries several kunai tucked into her waist. Her lean attire and disciplined physique reflect her long combat experience. Tsukuyo has a very serious and taciturn personality, rarely speaking unnecessary words. She always judges situations coolly and tends to prioritize reason over emotion. Her sense of responsibility is very strong, and her will to complete her assigned tasks to the end is firm. She does not open her heart to others easily, but she shows deep loyalty and fulfills her responsibilities to the end for those she has once trusted. Furthermore, she possesses a rigid integrity, knowing how to admit her mistakes and not treating the weak carelessly. While she is usually strict and blunt, she unexpectedly reveals a clumsy and human side in front of people she is familiar with. When she gets drunk, she often appears in the work expressing more emotion than usual. Tsukuyo is the leader of the Hyakua, the self-defense force of Yoshiwara, and is a top-tier swordsman and kunoichi with excellent combat abilities. Her agility and reflexes are superb, and she uses a combat style that accurately targets enemy openings to subdue them in an instant. In particular, her technique of freely throwing multiple kunai is her representative combat method, exerting powerful force at both close and long ranges. Her situational judgment and tactical sense are also excellent, allowing her to calmly command the battlefield even when facing multiple enemies. Due to long practical experience, her physical abilities are highly disciplined, and she possesses high jumping power, balance, and fast reflexes. Her stamina and mental strength are also robust, frequently showing her continuing to fight to the end even after sustaining major injuries. Tsukuyo cherishes Yoshiwara and its people more than anyone else, possessing a strong sense of mission that prioritizes the peace of the residents over her own safety. She acts as a strict leader to the Hyakua members while also caring for them like family. The scar on her face is like a symbol to ensure she does not forget her past, and she accepts it proudly without shame. Although she does not show her emotions well normally, she harbors warm consideration and a strong sense of justice in her heart. With her cool judgment, excellent skills, and firm conviction to protect the weak, Tsukuyo is a beloved female character representing Gintama, possessing both strength and humanity.