In the present work, ZnGa₂O₄ was incorporated with g-C₃N₄ nanosheets to synthesize ZnGa₂O₄/g-C₃N₄ nanocomposites through the hydrothermal method. The morphologies of nanocomposites were characterized by TEM, XRD, and spectral and electrochemical methods, respectively. The nanocomposites exhibited greatly enhanced fluorescence with the maximum emission peak red-shifted from 380 nm to 450 nm. A strong cathodic electrogenerated chemiluminescence (ECL) signal of ZnGa₂O₄/g-C₃N₄ nanocomposites was obtained under neutral conditions, which was much stronger than those of pure materials. ECL resonance energy transfer (ECL-RET) occurred between ZnGa₂O₄/g-C₃N₄ and gold nanoparticle/graphene nanocomposites, resulting in an apparent decrease of the ECL signal. Based on this, a label-free ECL sensor for thrombin was fabricated. The sensor showed high sensitivity, wide linearity, and good selectivity for the detection of thrombin in the range from 1.37 fM to 27.4 pM with a detection limit of 0.55 fM (3σ). The proposed method was applied to detect thrombin in serum samples with satisfactory results. This work revealed a new role of spinel-type semiconductor oxide nanomaterials, which will provide more ECL systems for the fabrication of biosensors.