The accurate measurement of full six degrees-of-freedom (6DOFs) knee joint kinematics is prohibited by soft tissue artifact (STA), which remains the greatest source of error. The purpose of this study was to present and assess a new femoral clamp to reduce STA at the thigh. It was hypothesized that the device can preserve the natural knee joint kinematics pattern and outperform a conventional marker mounted rigid cluster during gait. Six healthy subjects were asked to walk barefoot on level ground with a cluster marker set (cluster gait) followed by a cluster-clamp-merged marker set (clamp gait) and their kinematics was measured using the cluster method in cluster gait and the cluster and clamp methods simultaneously in clamp gait. Two operators performed the gait measurement. A 6DOFs knee joint model was developed to enable comparison with the gold standard knee joint kinematics measured using a dual fluoroscopic imaging technique. One-dimensional (1D) paired t-tests were used to compare the knee joint kinematics waveforms between cluster gait and clamp gait. The accuracy was assessed in terms of the root-mean-square error (RMSE), coefficient of determination, and Bland–Altman plots. Interoperator reliability was assessed using the intraclass correlation coefficient (ICC). The result showed that the femoral clamp did not change the walking speed and knee joint kinematics waveforms. Additionally, clamp gait reduced the rotation and translation errors in the transverse plane and improved the interoperator reliability when compared to the rigid cluster method, suggesting a more accurate and reliable measurement of knee joint kinematics.