This study presents a simple electrochemical approach for preparing a poly(L-serine) film-modified glassy carbon electrode, which responds quickly and sensitively during the simultaneous determination of acetaminophen and ofloxacin in prepared, environmental, and pharmaceutical samples. The prepared electrode exhibited catalytic activities and promoted the oxidation of acetaminophen and ofloxacin. Acetaminophen and ofloxacin showed linear responses between 1.0 × 10(-5) and 1.0 × 10(-4) mol/L and their limits of detection were 1.2 × 10(-7) and 1.6 × 10(-7) mol/L, respectively. The average recoveries (± relative standard deviations) of acetaminophen and ofloxacin were 96.8 ± 3.5 % and 97.6 ± 3.2 %, respectively, indicating that the prepared electrode and detection method are very accurate and reproducible for the simultaneous determination of acetaminophen and ofloxacin.