Methylene urea (MU) is a kind of slow-release fertilizer. The application of MU can increase nitrogen use efficiency, reduce environmental impacts and the labor intensity of farmers. This study investigated a rapid, simple and practical way to research MU biodegradation. The activated sludge after sterilization was used as the carrier for microbial growth and the soil with the MU-degrading microorganisms was added as inoculum. By adding glucose as the carbon source and MU as the nitrogen source, after 10–15 days of aerobic training at room temperature, the activated sludge with large amounts of MU-degrading microorganisms was used as research material. The hydrolysis of the urea released from dimethylenetriurea (DMTU) degradation was inhibited by N-(n-Butyl)thiophosphoric triamide (NBPT). The results showed that the nitrogen release process of DMTU is as follows: 1) the dissolved DMTU was firstly absorbed by microorganisms and then decomposed into urea, ammonium and formaldehyde by intracellular enzyme; 2) the urea and the ammonium nitrogen from DMTU degradation were released into the environment; 3) the urea released is further hydrolyzed into ammonium and carbon dioxide by urease in the extracellular environment. Because the dissolved DMTU can be rapidly mineralized by microorganisms and the degradation rate of DMTU is a constant, the degradation rate of DMTU is independent of the concentration of reactants and determined by other factors.