[Objectives] The effect of the fourth-type urease inhibitors on soil microorganisms was investigated to provide a theoretical basis for the safety evaluation of slow-release urea containing Schiff base complexes urease inhibitors in agricultural production.[Methods] Using indoor constant temperature and humidity incubation, the soil urease activity and bacteria, fungi and actinomycetes were determined at different concentrations(0.1%, 0.5%, 1% of urea application) under the condition of new Schiff copper base urease inhibitors.[Results] ① When the inhibitor concentration was 0.5% and 0.1%, the effect on soil urease activity was not significant. When the concentration was 1%, the inhibitory effect on soil urease activity was the best, and the maximum inhibition rate was 40.8%, which played the role of moderate regulation. ② Soil bacteria, fungi and actinomycetes showed different sensitivity to urea hydrolysis, among which actinomycetes and fungi were more sensitive. The maximum inhibitory rate of urea hydrolysis on soil actinomycetes and fungi was 46.4% and 89.7%, respectively. In contrast, the hydrolysis of urea can promote the growth of bacteria, and the maximum promotion rate is 83.6%. ③ The fourth-type urea inhibition could promote the growth of soil bacteria, actinomyces and fungi. The maximum promotion rate of bacteria, actinomyces and fungi is 86.2%, 31.9% and 83.6%, respectively. Therefore, it can be concluded that the fourth-type urease inhibitors have little effect on actinomycetes, while have greater effect on bacteria and fungi.[Conclusion] The fourth-type urease inhibitor has a good inhibitory effect on soil urease activity and can promote the growth of soil bacteria, fungi and actinomycetes. The optimal application concentration of the fourth-type urease inhibitor is 1%.