In this work synthesis of water-dispersible reduced graphene oxide (rGO), decorated with gold nanoparticles (AuNPs), was carried out in the presence of poly(diallyldimethylammonium chloride) solution (PDDA). The as-prepared hybrid nanocomposite (Au/PDDA/rGO) was characterized using various surface, structural, and electrochemical analysis techniques, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and voltammetry. The electrochemical performance of Au/PDDA/rGO-modified glassy carbon electrode (GCE) was investigated by studying the voltammetric behavior with different concentrations of levofloxacin (LV). Under optimized experimental conditions, at the Au/PDDA/rGO/GCE it was found that the response is linearly proportional to the concentration of LV in the ranges of 1.0 x 10(-5)-2.0 x 10(-4) and 2.0 x 10(-4)-8.0 x 10(-4) mol L-1, with detection limit estimated to be 3.9 x 10(-6) mol L-1. The fabricated sensor was successfully applied for determination of LV in pharmaceutical tablets. Additionally, investigation of the interaction between LV and RNA molecules has also been carried out. (C) 2015 The Electrochemical Society. All rights reserved.