Objective To evaluate the reliability and validity of the three-dimensional motion capture analysis system COFT-Motion® in evaluating the active range of motion of upper limbs in healthy subjects, and provide objective basis for its clinical application. Methods From January to March 2022, healthy subjects were publicly recruited for enrollment. The evaluator applied the three-dimensional motion capture analysis system COFT-Motion® and angle ruler to evaluate the active range of motion of the shoulder joint (flexion, extension, adduction, abduction) and elbow joint (flexion, extension) in healthy subjects. Pearson correlation analysis was used to evaluate validity, and intraclass correlation coefficient (ICC) was used to evaluate reliability. Results There was a positive correlation between the measurements of COFT-Motion® and the angle ruler of the active range of motion of upper limbs in healthy subjects. The Pearson correlation coefficients for active range of motion of the shoulder joint (flexion, extension, adduction, abduction) and elbow joint (flexion, extension) were 0.913, 0.964, 0.961, 0.941, 0.864, and 0.919, respectively (P<0.05). The results of using COFT-Motion® by different evaluators to evaluate the active range of motion of healthy subjects’ shoulder joints (flexion, extension, adduction, abduction) and elbow joints (flexion, extension) showed that the ICC values were 0.892, 0.942, 0.961, 0.988, 0.989, and 0.928, respectively (P<0.05). The results of repeated evaluations by the same evaluator showed that the ICC values were 0.795, 0.916, 0.900, 0.868, 0.918, and 0.911, respectively (P<0.05). Conclusion The application of the three-dimensional motion capture analysis system COFT-Motion® in healthy subjects has good validity and reliability. However, due to the limited sample size and subject of the study, further promotion and application of the system still need to be further explored.