Detection of different cancers such as prostate cancer (PCa) in the initial stage is so important in biomedical research so, the early, definitive, and sensitive diagnosis of PCa is essential to avoid the worsening development of this disease. In this paper, we designed two simple and novel electrochemical biosensors (PCW/TGA/AuNPs/RGO/GCE and PCW/TGA/AuNPs/CHT/GCE). The designed biosensors were fabricated based on glassy carbon electrodes (GCE) modified by nanomaterials of reduced graphene oxide (RGO) and chitosan (CHT) and electrodeposited gold nanoparticles (AuNPs) at the self-assembled mono layers of thioglycolic acid (SA-TGA) linker for detection of DNA sequences of PCa. The biosensors surfaces were characterized using scanning electron microscope (SEM), and electrochemical techniques. The immobilization of the probe (PCW) and its hybridization with the complementary DNA (PCWC) was optimized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques in an aqueous solution. Under the optimized experimental conditions, wide linear range and low detection limit for prostate cancer (PCa) were determined by using PCW/TGA/AuNPs/RGO/GCE and PCW/TGA/AuNPs/CHT/GCE biosensors. The suggested biosensors were applied for determination of DNA sequences and showed excellent selectivity for discriminating the complementary sequences from various targets suchas non-complementary, one base-mismatch and three base mismatch sequences. It is promising that the designed biosensors can be applied for detection of prostate cancer mutation in the future. (C) 2016 Elsevier B.V. All rights reserved.