Soil microorganisms are soil nutrient biological driving factors, and nitrogen deposition can change its activity and biomass, which can break the soil nutrient cycling and dynamic equilibrium. The influence of nitrogen deposition on soil microbial biomass in tropical, subtropical and temperate virgin forest ecosystems is increasingly investigated, however, there are less reports from temperate natural secondary forests. We conducted field surveys in May (spring), July (summer) and September (autumn) of 2016 in the control test sample-Betula platyphylla-Populus davidiana natural secondary mixed forest, in which we simulated nitrogen deposition from 2006 in the Changbai mountains. The control trial was divided into three nitrogen-adding treatments:Control (CK 0 kg N hm^-2 a^-1), Low-nitrogen (LN 25 kg N hm^-2 a^-1) and High-nitrogen (HN 50 kg N hm^-2 a^-1). According to the soil layer (0-10 cm and 10-20 cm), we separately tested the soil microbial biomass carbon (MBC) and nitrogen (MBN), soil total carbon (TC), nitrogen (TN) and phosphorus (TP), pH, soil dissolved organic carbon (DOC) and nitrogen (DON). The results showed that, 1) the soil pH significantly declined under the effect of nitrogen deposition. The TC and TN in the upper layer of the soil were less impacted by nitrogen deposition, and nitrogen deposition significantly increased the TC and TN content of the deeper soil. Nitrogen deposition also increased the TP content of the soil in spring and summer. In autumn, the LN treatment inhibited TP. The impact of nitrogen deposition on DOC and DON were not significant. 2) The upper soil MBC showed a decreasing trend in spring and autumn, and the deeper soil increased first and then decreased. HN had an inhibitory effect on MBC, and LN promoted MBC in the deeper soil. The soil MBN showed a decreasing trend from spring to autumn, and the difference between the upper and deeper soil MBN was significant. Nitrogen treatment promoted MBN in spring and autumn, and had an inhibitory effect in summer. Nitrogen deposition also reduced MBC/MBN in spring and autumn and increased MBC/MBN in summer soil. 3) The effects of nitrogen treatment, seasonal variation and soil depth on MBC and MBN were significant, and had significant interacting effects. In summary, the effect of long-term nitrogen deposition on soil microbial biomass in the growing season has seasonal differences and is influenced by the depth of the soil layer. Future research should pay attention to the interannual change, but also investigate the difference between spatiotemporal dynamics and nitrogen deposition.