A rapid microwave-hydrothermal method has been developed to prepare monodisperse colloidal carbon nanospheres from glucose solution, and gold nanoparticles (AuNPs) are successfully assembled on the surface of the colloidal carbon nanospheres by a self-assembly approach. The resulting AuNP/colloidal carbon nanosphere hybrid material (AuNP/C) has been characterized and is expected to offer a promising template for biomolecule immobilization and biosensor fabrication because of its satisfactory chemical stability and the good biocompatibility of AuNPs. Herein, as an example, it is demonstrated that the as-prepared AuNP/C hybrid material can be conjugated with horseradish peroxidase-labeled antibody (HRP-Ab2) to fabricate HRP-Ab2-AuNP/C bioconjugates, which can then be used as a label for the sensitive detection of protein. The amperometric immunosensor fabricated on a carbon nanotube-modified glass carbon electrode was very effective for antibody immobilization. The approach provided a linear response range between 0.01 and 250 ng mL−1 with a detection limit of 5.6 pg mL−1. The developed assay method was versatile, offered enhanced performances, and could be easily extended to other protein detection as well as DNA analysis.