Graphene oxide (GO) has been widely used in many applications due to its excellent properties. Given the extensive production and use of this nanomaterial, its release into the environment is inevitable. However, little is known about the effects of GO on microbial nitrogen transformation and the related processes after GO enters the soil environment. The present study showed that GO significantly reduced soil microbial biomass and caused a decline in microbial diversity after the soils were subjected to various GO concentrations (10, 100, and 1000 mg kg) for 4 months. Among them, the abundances of nitrogen transformation related bacteria such as Firmicutes, Nitrospirota, Proteobacteria, Planctomycetota, and Cyanobacteria were significantly decreased with GO incubation. Among the enzymes that are related to nitrogen transformation, nitrate reductase was the most sensitive even at low concentrations of GO, followed by ammonia monooxygenase and urease, which were reduced by 13-31%, 5-26%, and 9-19% respectively, than those of the control. We found that high concentrations of GO significantly increased the retention of soil urea by 32-59%, and the contents of ammonium and nitrate were 22-28% and 55-69% lower compared to those of the control, respectively. Moreover, the response of most of the indicators in the above process to multilayer GO was more significant than that to single layer GO. Overall, this study provides new insights into the comprehensive understanding of GO's impacts on the soil nitrogen cycle.