The excessive use of levofloxacin (LEV) can lead to significant environmental pollution, while also damaging human health. The accurate detection of its concentration is therefore essential to protect both the environment and human health. Herein, we report the simple synthesis of α-zirconium phosphate supported on nitrogen-doped graphene nanosheets (α-ZrP/NG) and its application in the voltammetric determination of trace LEV in real samples. The surface morphology, microstructure, and electrochemical properties of α-ZrP/NG were investigated by microscopic and spectroscopic techniques. The analytical parameters, including the accumulation potential, accumulation time, loading volume, and buffer pH, were optimized to improve the electrocatalytic sensing performance of α-ZrP/NG. Owing to the synergetic interaction between α-ZrP and NG, the proposed sensor exhibited an excellent electrocatalytic activity for the electrical oxidation of LEV. In the range of 0.01−5.0 μM, the anodic peak current of LEV increased linearly with an increase in the LEV concentration, and the limit of detection was 2.6 nmol L −1 . Finally, the LEV levels in wastewater, human serum, milk, and eye drop samples were determined by using the α-ZrP/NG system combined with a glassy carbon electrode to give satisfactory recoveries (98.48−103.2%), indicating that the prepared sensor demonstrates promise for use in environmental, food, and drug monitoring applications.