Abstract: Electrocatalytic glycerol oxidation reaction (GOR) enables the conversion of glycerol, a by-product of the biodiesel industry, into high-value-added chemicals such as lactic acid, and is regarded as a potential alternative to the kinetically sluggish anodic oxygen evolution reaction in water electrolysis. However, commonly used noble-metal catalysts suffer from high cost, whereas non-noble-metal catalysts still exhibit insufficient intrinsic activity and limited product selectivity. A series of CuNi alloy catalysts, including CuNi, CuNi−10, CuNi−20, and CuNi−30, are prepared by electrodeposition combined with a chemical etching strategy, and the surface defect structure and electronic state of the catalysts are regulated by varying the etching time. Electrochemical results show that the etching treatment changes the GOR behavior of CuNi alloys, among which CuNi−20 exhibits relatively high glycerol oxidation activity. At 1.25 V vs. RHE, CuNi−20 achieves a lactic acid selectivity of 84.23% and a lactic acid yield of 1.52 mg/(h·cm²), and maintains stable operation during a 40 h chronoamperometric test. Structural characterization and mechanistic analysis indicate that appropriate etching introduces defect sites on the catalyst surface, promotes the exposure and formation of high-valence Ni-based active species, and modulates the electronic interaction between Cu and Ni, thereby affecting the adsorption behavior of reactants and intermediates as well as the product distribution. Reaction pathway analysis shows that glycerol oxidation to the dihydroxyacetone intermediate is promoted over CuNi−20 under alkaline conditions, followed by conversion to lactic acid through a rearrangement process. These results indicate that the synergistic effect between surface defects and high-valence metal species is an important factor in regulating the GOR activity and lactic acid selectivity of CuNi alloys.