The reactions of glyoxal (CHO)₂) with amines in cloud processes contribute to the formation of brown carbon and oligomer particles in the atmosphere. However, their molecular mechanisms remain unknown. Herein, we investigate the ammonolysis mechanisms of glyoxal with amines at the air-water nanodroplet interface. We identified three and two distinct pathways for the ammonolysis of glyoxal with dimethylamine and methylamine by using metadynamics simulations at the air-water nanodroplet interface, respectively. Notably, the stepwise pathways mediated by the water dimer for the reactions of glyoxal with dimethylamine and methylamine display the lowest free energy barriers of 3.6 and 4.9 kcal ⋅ mol⁻¹, respectively. These results showed that the air-water nanodroplet ammonolysis reactions of glyoxal with dimethylamine and methylamine were more feasible and occurred at faster rates than the corresponding gas phase ammonolysis, the OH+(CHO)₂ reaction, and the aqueous phase reaction of glyoxal, leading to the dominant removal of glyoxal. Our results provide new and important insight into the reactions between carbonyl compounds and amines, which are crucial in forming nitrogen-containing aerosol particles.