Purpose: A novel portable system has been used to evaluate spatial knee movement, but its accuracy and repeatability is not known. The aim of this study was to investigate the accuracy and repeatability of the measurement. Methods: Ten healthy participants were included, and the knee motion trajectory during walking were assessed. Six evaluations were conducted (three days by two raters) for each participant. The motion parameters at the key points and the range of motion were statistically analyzed. Intraclass correlation coefficients (ICC), standard error of the measurement (SEM), and the Bland–Altman method were used. Results: For intra-rater repeatability, 1) the ICC values range from 0.75 to 0.9 for rotations; and 0.64 to 0.96 for translations. Among the ICC values, 100% of rotations and 90% of translations were not less than 0.70; 2) among the SEM values, 100% of rotations were not more than 5°, while 73.3% of translations were less than 3 mm. For inter-rater repeatability, 1) the ICC values range from 0.68 to 0.99 for rotations; and 0.57 to 0.93 for translations. Among the ICC values, 95.6% of rotations and 82.2% of translations were not less than 0.70; 2) among the SEM values, 100% of rotations were not more than 5°, and 48.9% of translations were less than 3 mm. The Bland–Altman plots showed good agreement for intra- and inter- repeatability. Conclusions: The results indicated that the accuracy and repeatability of the measurement were acceptable, except for the inter-rater repeatability for translation. This may help researchers and physicians better interpret the measurement data.