The sensitive and specific detection of tumor biomarkers is crucial for early diagnosis and treatment of malignant melanoma. Immunoassay with a simple sensing interface and high sensitivity is highly desirable. In this work, a simple electrochemical immunosensor based on a chitosan/reduced graphene oxide (CS-rGO) nanocomposite was developed for sensitive determination of an S-100B protein, a tumor marker of malignant melanoma. CS-rGO nanocomposite were prepared by chemical reduction of graphene oxide in the presence of chitosan and modified on glassy carbon electrode (GCE) to provide a biofriendly, conductive, and easily chemically modified matrix for further immobilization of antibodies. Anti-S-100B antibodies were grafted onto the chitosan molecules to fabricate the immunorecognition interface by a simple glutaraldehyde cross-linking method. Electrochemical determination of S-100B was achieved by measuring the decreased current signal of solution phase electrochemical probes, which originated from the increased steric hindrance and insulation caused by the formation of antigen-antibody complexes at the electrode interface. Due to the good conductivity, high surface area, excellent biocompatibility, and good film-forming ability of CS-rGO, the constructed immunosensor exhibited good stability, high selectivity and sensitivity, a wide dynamic range from 10 fg mL(-1) to 1 ng mL(-1) and a low limit of detection of 1.9 pg mL(-1) (S/N = 3). Moreover, the sensor was also applicable for the sensitive detection of S-100B protein in real human serum samples.