A glassy carbon electrode (GCE) modified with a chemically reduced graphene oxide and chitosan (CRGO/CS) composite film was constructed and used to determine 5-fluorouracil using cyclic, staircase and square wave voltammetric techniques. The synthesized graphene oxide material was characterized by XRD, FTIR and UV spectroscopic techniques. The surface morphology of the CRGO/CS film was characterized using atomic force microscopy. The oxidation potential shifted negatively compared to GCE, indicating that the CRGO/CS modified electrode had obvious catalytic activity for the 5FU oxidation. The effects of pH, scan rate and excipients were studied. The sensor showed a good detection limit and it was successfully applied to determine 5FU in pharmaceutical and human biological samples.