Objective To investigate gait characteristics of both lower limbs in female patients before and after unilateral total knee arthroplasty (TKA). Methods Gait parameters of 10 female patients were compared in both lower limbs, and compared to those of 10 healthy subjects based on three-dimensional motion analysis system and independent Student’s t test. Results The preoperative stance phase, peak adduction angle, adduction-abduction range of motion (ROM) and peak flexion moment of the uninvolved knee were significantly higher than those of the involved side (P<0.05), while postoperative gait parameters were similar in both sides. The patients had significantly lower speed and stride length, longer double support time than the control group before TKA (P<0.05). Moreover, the involved knee demonstrated significantly lower peak extension and adduction angles, adduction-abduction ROM, peak flexion and abduction moments, while higher peak adduction moment, and the uninvolved knee revealed significantly lower peak extension angle, higher peak flexion and adduction moments than the control group before TKA (P<0.05). However, peak extension angles and adduction moments in both sides were still different with those of the control group after TKA (P<0.05). Conclusions Patients had pain relief and improved gait in both sides after TKA. However, there were gait differences compared to the control group, because of the deficits in joint function, such as muscle strength and proprioception, and pathological gait and posture still existing. Therefore, patients should enhance muscle strength of the involved limb, and take gait correction training. Female patients should also reduce walking speed and improve proprioception. The conclusion presents important references for pre-operative evaluation, prevention and post-operative recovery for female patients.