Highly sensitive and enzyme-free detection of melanoma adhesion molecule antigen (CD146) remains a challenge in clinical diagnosis. The prepared immunosensor, based on amination graphene (GS-NH2) and mesoporous nano-Co3O4 sheet combined with gold nanoparticles (Au/Co3O4), exhibited significantly increased electron transfer, high sensitivity and stability to CD146. Au/Co3O4 can increase the contact surface between the antibody and Au nanoparticles attached on Co3O4 than mesoporous Co3O4 only. And the mesoporous Co3O4 nanosheet can capture more biomolecules to enhance the sensitivity due to the large effective specific area. Amperometric i-t curve and electrochemical impedance spectroscopy (EIS) were used to characterize the recognition of CD146. This novel immunosensor, works well over a broad linear range of 0.01-15ng/mL, with a low detection limit of 3.4pg/mL (S/N=3). The immunosensor was evaluated for the determination of human serum sample, and received a satisfactory result. The developed immunosensor provides a promising approach for clinical research and diagnostic applications. Copyright © 2014 Elsevier B.V. All rights reserved.