A novel biomolecular immobilization strategy based on gold nanoparticles-polydopamine-thionine-graphene oxide (Au-PDA-THi-GO) nanocomposites was used to develop a highly sensitive label-free electrochemical immunosensor for the detection of alpha-fetoprotein (AFP). The morphologies and characteristics of the as-prepared Au-PDA-THi-GO nanocomposites were investigated by using scanning electron microscope (SEM), atomic force microscopy (AFM), energy dispersive X-ray spectrometer (EDX), UV-vis spectroscopy and electrochemical methods, respectively. The as-prepared Au-PDA-THi-GO nanocomposites not only provided a favorable microenvironment to maintain the activity of the immobilized biomolecules due to the excellent biocompatibility of PDA, but also increased the loading capacity of the biomolecules due to the large surface area of nanocomposites, and the present of gold nanoparticles enhanced the conductivity and charge-transport properties of the composites. Under optimal conditions, the present immunosensor exhibited a wide linear range from 0.1 to 150.0 ng mL(-1) for detection of AFP (as a model analyte), with a low detection limit of 0.03 ng mL(-1) (at signal to noise ratio of 3). The immunosensor exhibited high selectivity, long-term stability and good reproducibility. (C) 2013 Elsevier B.V. All rights reserved.