Chiral identification has significant research value in fields such as food and medicine, mainly due to the specific expression of biomolecular enantiomers. This study focused on the enantioselective recognition problem of L-/D-threonine and developed a novel chiral electrochemical sensor. The specific approach was to modify L-proline onto the surface of UiO-66-NH₂ to form the UiO-66-NH₂ -L-Pro complex. The preparation process adopted solvothermal technology combined with ultrasonic refining. The FT-IR results confirmed that both the Zr-O bonding performance and the framework coordination degree were significantly optimized under this ideal condition, and the differential pulse voltammetry (DPV) was used to evaluate the chiral sensing characteristics of this material. The L-threonine and D-threonine had recovery rates of 21.0–106.3 and 45.5–97.8 in percentage, which implied the applicability of the sensor in complex bio-samples. In a straightforward and efficient way, this modification method increases interface exposure and response efficiency. The sensor has great potential for use in food safety monitoring, pharmaceutical quality control, and clinical diagnostics. This strategy will be expanded to other chiral biomolecules in future research, and sensor stability and sensitivity will be improved for real-world applications.