Adriamycin (ADM)-coated silica microspheres as a label for the sensitive detection of a cancer biomarker alpha-fetoprotein (AFP) was reported. Silica microspheres (SiO2 MSs) were employed as the carrier for the immobilization of gold nanoparticles (Au NPs), secondary antibody (Ab2) and ADM (denote: ADM@Au NPs@SiO2 MS/Ab2) as labels. In the presence of AFP, the labels were captured on the surface of the Au NP-reduced graphene oxide (rGO) (Au NP-rGO) nanocomposites to form a sandwich structure vs. the specific recognition of antibody-antigen. In a pH 7.4 phosphate buffer solution, a well-defined peak of ADM at about -0.70 V (vs. SCE) was recorded via differential pulse voltammetry, the peak intensity of which was related to the concentration of AFP. Under optimal experimental conditions, the immunoassay exhibited a wide linear range (0.5 pg mL-1 to 75 ng mL-1) and low limit of detection (0.17 pg mL-1). Further, the immunoassay was evaluated for serum samples, which gave satisfactory results.