Developing functionalized nanomaterials with strong chemiluminescence (CL) properties is highly significant for ultrasensitive bioanalysis. Here, we report chitosan (CS), luminol, and Co2+-functionalized flower-like gold nanoparticles (Co2+/CS/Lum/AuNFs) with strong CL for the label-free sensing of the HCV core protein (HCVcp). The Co2+/CS/Lum/AuNFs exhibited a greatly enhanced CL emission at around 425 nm, which is 50 times stronger than that of CS/Lum/AuNFs, and is superior to other commonly reported CL nanomaterials. The HCVcp aptamer (HCVcp-apt) further functionalized the surface of the Co2+/CS/Lum/AuNFs through electrostatic interactions blocked the Co2+ catalytic site, depressing the CL. Owing to the high affinity of HCVcp for the HCVcp-apt, the presence of HCVcp predominated its binding and effectively separated the HCVcp-apt from the surface of the Co2+/CS/Lum/AuNFs, so that the CL intensity was significantly enhanced. As the results showed, the HCVcp-apt/Co2+/CS/Lum/AuNFs were successfully used to detect the HCVcp in human serum samples with a linear range from 0.50 ng mL-1 to 1.00 μg mL-1, a detection limit of 0.16 ng mL-1 and an excellent selectivity over other analogs. The strategy is universal for the development of the ultrasensitive detection of other proteins in the field of early disease diagnostics.