In this study, barley leaves were used as biomass precursor to prepare the ZnCl2 activated barley leaves derived carbon by the carbonization under N2 atmosphere at high temperature (ZnAC). The gold nanoparticles were synthesized in situ on the surface of ZnAC (AuNPs/ZnAC). The as-prepared AuNPs/ZnAC composite was characterized by SEM, Raman spectroscopy, XRD et. al. The AuNPs/ZnAC composite was drop-cast onto the surface of a glassy carbon electrode to fabricate an electrochemical sensing platform for the simultaneous determination of acetaminophen (AC) and levofloxacin (LEV). The results of electrochemical experiments showed that ZnAC and AuNPs had a synergetic electrocatalytic effect on the oxidation of AC and LEV. The oxidation peak potentials of AC and LEV were at 0.57 and 1.02 V, respectively. The proposed sensor allowed the simultaneous quantification of AC and LEV two linear intervals: in the ranges of 0.1–15 and 15–300 μM for AC and 0.1–20 and 20–300 μM for LEV with the detection limits of 0.02 and 0.03 μM, respectively. In addition, the sensor has the advantages of low cost, high sensitivity, simple preparation and environmental friendliness, as well as good selectivity, stability and reproducibility. We measured AC and LEV in artificial urine, tap water, and lake water, and the recoveries were between 93 % and 109 %, indicating that the sensor has good application prospects in biomedicine and environmental protection.