Electrochemiluminescence (ECL) nanomaterials are usually deposited compactly on the surface of electrodes, which may cause poor mass transfer of reactants, thereby resulting in low ECL efficiency. In this work, we developed a novel kind of luminescent material denoted as C-Au-luminol nanospheres (C-Au-Lum NSs) by high dispersion of luminophores on porous carbon nanospheres (PCNSs). C-Au-Lum NSs were facilely prepared by the in situ reduction of chloroauric acid with the luminescent reagent luminol (Lum) on the nano-pores of PCNSs. Plenty of luminescent Au-Lum NPs were dispersedly concentrated inside the numerous pores and hollow interiors of PCNSs, effectively increasing the mass transfer of reagents and accelerating the electron transport inside the porous nanospheres. This greatly improved the availability of luminophores and endowed C-Au-Lum NSs with excellent ECL emission. After further integrating with enzymatic circulation and strand displacement, an ultrasensitive ECL biosensor was achieved for the ultrasensitive detection of an important tumor biomarker, mucin1. The logarithmically linear range from 0.1 pg mL-1 to 1 ng mL-1 with the detection limit of 47.6 fg mL-1 (S/N = 3) was achieved, demonstrating the superior performance of C-Au-Lum NSs. This work would provide new ideas for the construction of high-performance ECL sensing platforms for diverse applications.