Microbial residue nitrogen can indicate soil quality and is crucial for soil nitrogen retention and supply. However, it is still unclear how the dynamic changes in soil microbial residue nitrogen affect crop nitrogen uptake in agricultural practice. Based on a long-term wheat-maize rotation experiment with different nitrogen application rates (150 kg N ha⁻¹, 200 kg N ha⁻¹, and 300 kg N ha⁻¹), ¹⁵N-labeled nitrogen fertilizer was applied during the wheat season to track the dynamics of soil microbial residue nitrogen and its mediated fertilizer nitrogen. The results showed that nitrogen addition was beneficial to the accumulation of microbial residue nitrogen (mainly fungal microbial residue nitrogen). Its contribution rate to soil total nitrogen is 54.87–56.55%, and the fertilizer nitrogen allocated to it accounts for 27.10–47.50% of the remaining fertilizer nitrogen in the soil. Ultimately, 6.77–10.24% of the nitrogen fertilizer applied remained in the soil as microbial residue nitrogen. In addition, microbial residue nitrogen is mainly mineralized during the jointing and filling stages of wheat. In a word, the accumulation and mineralization of soil microbial residue nitrogen during the application of 200 kg N ha⁻¹ better guaranteed the uptake of nitrogen by wheat, which provided a reliable basis for guiding farmland fertility improvement and nitrogen fertilizer reduction in the North China Plain.