Aging-related loss of muscle strength with a cumulative loss of muscle mass results in functional impairment and risk of falling. Moreover, knee extensor muscle weakness is associated with a higher risk of knee osteoarthritis in both females and males. The variability of electromyography (EMG) signals in males and females varied in each gait phase. The characteristics of knee EMG during a gait cycle in female and male elderlies have not been clarified clearly. The purpose of this study was to determine the differences in knee muscle between female and male elderlies during overground walking. Five healthy females and five healthy males with ages over 60 years old were recruited for this study. The participants performed 3 trials of walking back and forth at a comfortable gait speed on a 10-meter walkway. The surface EMG signals were recorded on two muscles in each leg, comprising the rectus femoris (RF), and the long head of biceps femoris (BF) during the entire gait cycle. The knee muscle activity was investigated considering the right and left sides together in the root mean square (RMS) and waveform length (WL). The asymmetry index (ASI) of the knee muscle between the right and left sides in female and male elderlies was evaluated in the mean absolute value (MAV) and log detector (LOG). The characteristics of EMG signals were evaluated in the stance and swing phases of the gait cycle. This study found that RMS and WL of RF and BF were not different between elderly females and males during the entire gait cycle. Asymmetry index of MAV of BF (male 47.61 ± 27.71 vs. female 16.45 ± 11.68) and LOG of RF (male 10.07 ± 1.86 vs. female 4.54 ± 4.71) and BF (male 14.80 ± 8.77 vs. female 5.04 ± 3.31) in males was higher than females during a swing phase. This pilot study provides useful information to characterize knee muscle activity in elderlies. The knee muscle activities between female and male elderlies were not much different in both gait phases. Interestingly, the asymmetry between limbs of the knee muscles during the swing phase in elderly males was higher than in elderly females although they are healthy.