In this study, we fabricated a label-free electrochemical immunosensor for sensitive and selective detection of tumor marker p53. This immunosensor was based on chitosan/carbon black composite (Chitosan-CB) layer coated ITO electrode. This composite was utilized for enhancement of the conductivity of the immunosensor. Anti-p53 antibodies were captured on the modified ITO electrode through the cross-linking of chitosan and glutaraldehyde. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were utilized for electrochemical characterization of the proposed immunosensor. Moreover, the biosensor construction steps were monitored by using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The immobilization of anti-p53 antibodies on the electrode surface was investigated by using Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. The change in impedance which formed during the specific interaction between anti-p53 antibody and p53 antigen was used to detect p53. Under optimized experimental conditions, the fabricated immunosensor had a wide linear range of 0.01-2 pg/mL and low detection limit of 3 fg/mL. The fabricated immunosensor had good sensitivity, stability and repeatability. Furthermore, it was successfully applied to analyze p53 in human serum.