Proprioception plays an important role in joint stability, and ankle sprains usually happen involving plantarflexion, internal rotation, and inversion. However, ankle 3D movement proprioception has never been measured in weight-bearing. Accordingly, the active 3-dimensional movement extent discrimination apparatus (AMEDA-3D) was developed and its reliability and validity were investigated. A total of 58 subjects volunteered for this trial, with 12 subjects with chronic ankle instability (CAI) and 12 healthy controls in the test-retest reliability study. There were 17 subjects with CAI and 17 healthy controls in the validity study. An intraclass correlation coefficient (ICC) and Minimum Detectable Change at the 90% confidence interval (MDC) were computed. AMEDA-3D scores were analysed by independent samples t-tests, and Youden's index was used to calculate the optimal AMEDA-3D cut-off for discriminating individuals with CAI. Pearson's correlation analysis was used to explore the relationship between AMEDA-3D scores and Y Balance Test (YBT), Time In Balance Test (TIB), and Cumberland Ankle Instability Tool (CAIT) scores. The main results were as follows: (1) The ICC value of AMEDA-3D scores was 0.817 (95% CI = 0.452-0.945) in CAI subjects. (2) The AMEDA-3D proprioceptive area under the curve score used to discriminate CAI subjects from healthy controls was 0.778, with a sensitivity of 94% and a specificity of 82%. (3) AMEDA-3D proprioceptive scores were moderately correlated with CAIT scores (r = 0.58 and p < 0.001), YBT (r = 0.47 and p = 0.005), and TIB (r = 0.68 and p < 0.001). Our findings suggest that the AMEDA-3D tool shows good reliability and validity for clinical assessment of proprioceptive deficits associated with CAI. Improved ankle 3D motor proprioception may positively impact subjects' balance control, self-rated symptoms, and function.