A polymetallic sulfide of Cu2S/Ni3S2 is assembled in situ on nickel foam (NF), serving as a self-supported electrode and electrocatalyst for non-enzymatic glucose sensing. Benefiting from interfacial synergistic effects, the Cu2S/Ni3S2/NF composite exhibits outstanding electrocatalytic capability toward glucose oxidation, with a high sensitivity of 22 750 µA mM−1 cm−2, a low limit of detection (LOD) of 0.51 µM, a broad linear detection range of 1 µM–1 mM, and good selectivity against interfering species. Density functional theory (DFT) calculations of the Cu2S/Ni3S2 heterojunction are performed to reveal the density of states (DOS) and charge density difference. Effective interfacial electron transfer between Cu2S and Ni3S2 modulates the electronic structure, thereby enhancing electrocatalytic activity. The optimal d-band centers (dbc) of Cu and Ni in Cu2S/Ni3S2 are beneficial for the promotion of glucose sensing sensitivity. This work provides an efficient strategy for fabricating polymetallic sulfides for application in next-generation non-enzymatic glucose sensing.