This study aimed to explore the effects of fatigue on the balance and ankle proprioception during drop landing of individuals with chronic ankle instability (CAI). A total of 35 participants with unilateral CAI and 35 healthy participants participated in this study. A static balance test, dynamic balance test, and ankle proprioception test were conducted before and after fatigue. Fatigue was induced with turn back runs and vertical jumps protocol. Sway distance of the center of pressure (COP), root mean square of the COP (RMS), total excursions (TOTEX), mean velocity (MVELO), 95% confidence ellipse area of the COP movements (95% AREA), Normalise Reach Distance in the anterior (ANT), posteromedial (PM), and posterolateral (PL) directions, and the area under the curve (AUC) were calculated and analyzed. There were significant group by fatigue interactions for static balance variables, normalise reach distance in the PM and PL directions, and AUC. Fatigue reduced balance and ankle proprioception in individuals with CAI. After fatigue, static and dynamic balance and ankle proprioception during drop landing were significantly worse in the CAI group than in the control group. Fatigue had a significant negative effect on balance and ankle proprioception in CAI patients. Therefore, fatigue may be an important factor causing repeated ankle sprain in CAI patients.