To develop the sensitive and selective levofloxacin sensors with simple preparation process, the novel poly(p-aminobenzene sulfonic acid)-reduced graphene oxide (poly(p-ABSA)-rGO) composite film-based electrochemical sensor was facilely and rapidly prepared by one-step electrochemical method without the expensive and troublesome electrode modification procedure. The poly(p-ABSA)-rGO composite film was found to possess remarkable catalytic activity on the oxidation of levofloxacin. Electrocatalytic performance of the as-prepared sensor could be dramatically improved by optimizing the synthesis conditions of composite film. Based on the synergetic effect of organic conductive polymer poly(p-ABSA) and rGO, the proposed sensor exhibited excellent electrocatalytic activity of levofloxacin, high sensitivity, low detection limit (0.12 mu M), wide linear range and excellent selectivity. Further, the fabricated sensor was successfully applied onto the determination of levofloxacin in real human urine samples and showed good reliability for real samples assay. Based on the simple construction process and excellent electrocatalytic performance, the proposed poly(p-ABSA)-rGO film-based sensor demonstrated the potential for the extensive applications on electroanalysis and bioelectrocatalysis.