In this paper, a linker-free connected graphene oxide/Au nanocluster (GO-Au NCs) composite was prepared under sonication through electrostatic interactions, and characterized by transmission electron microscope (TEM), atomic force microscope (AFM), ultraviolet-visible (UV-vis) and FT-IR spectrum. The morphological and structural characterizations evidence that the Au NCs can be efficiently decorated on the GO. The electrochemical investigations indicated that GO-Au NCs composite has an important role in the electrocatalytic activity towards the oxidation of I-cysteine (CySH). The GO-Au NCs composite modified eletrode shows a large determination range from 0.05 to 20.0 _mol/L, a remarkably low detection limit is 0.02 _mol/L and low oxidation potential (+0.387). It was found that metal ions, carbohydrates, nucleotide acids and amino acids had no distinct effect on the determination of I-cysteine. In addition, the sensor has some important advantages such as simple preparation, fast response, good stability and high reproducibility. The direct determination of free reduced and total CySH in human urine samples has been successfully carried out without the assistance of any separation techniques.