Reduced knee flexion is a leading feature of post-stroke gait, but the causes have not been well understood. The purpose of this study was to investigate the relationship between the knee flexion and the lower-limb muscle activation within the stance-to-swing phases of gait cycle in the post-stroke hemiplegic patients. Ten stroke patients and 10 age- and gender-matched healthy subjects participated in the experiment. The lower-limb kinematic signals and the surface electromyography (sEMG) signals of the left and right rectus femoris (RF), biceps femoris (BF) and lateral gastrocnemius (GS) were recorded during walking. The angle range (AR) of knee flexion, the root mean square (RMS) and the mean frequency (MNF) of sEMG signals were calculated from the terminal stance (TSt) to the initial swing (ISw) phases of gait cycle. Stroke patients showed lower bilateral AR of knee flexion and lower RMS of GS on the paretic side, but higher MNF of RF on the non-paretic side compared with the controls. Within the stroke patients, significant differences were found between their paretic and non-paretic limbs in the AR of knee flexion, as well as in the RMS and MNF of GS ( p < 0.05). Regression analysis showed that the RMS of BF, MNF of BF and MNF of GS explained 82.1% of variations in AR of knee flexion on paretic side ( r 2 = 0.821). But the RMS and MNF of all the muscles (including the RF, GS and BF) could explain 65.6% of AR of knee flexion variations on the non-paretic side ( r 2 = 0.656), and 45.2% of variations for the healthy subjects ( r 2 = 0.452). The reduced knee flexion during gait was associated with altered magnitude and frequency of muscle contractions and with simplified muscle synergy in the post-stroke hemiplegic patients. Identifying the muscles that are responsible for knee stiffness may facilitate improvement of rehabilitation strategy for post-stroke gait.