It is widely known that homocysteine (Hcy) is associated with the pathogenesis of many clinical diseases, and its quantitative detection can help early treatment of related diseases. The selection of materials with excellent properties is important for the construction of electrochemical sensing platforms and quantitative detection of Hcy. In this study, two nanocomposites, Au nanoparticles/hollow carbon spheres (AuNPs@HCS) and Au nanoparticles/solid carbon spheres (AuNPs@SCS), were prepared first. Then their morphology, structure and electrochemical behavior were investigated. And the comparison found that the HCSs have a large electroactive surface area and outstanding conductivity. Thanks to the catalytic properties of AuNPs and the synergistic effect of HCSs, AuNPs@HCS/GCE showed excellent electrochemical sensing performance for a wide range of Hcy concentrations with a detection limit down to 4.44 nM (S/N = 3). Moreover, the electrochemical mechanism of Hcy on Au@HCS/GCE is discussed in detail. Finally, in the actual serum sample assay, the sensor is used to detect Hcy directly and the recovery rate is satisfactory. This work may provide a valid method for the quantitative determination of Hcy in the future.