The direct-sown cotton after wheat harvest (DSCWH) cropping system has attracted wide attention due to reduced labor inputs compared to transplanting. However, the management strategy of slow-release nitrogen is unclear in such a system. This study aims to investigate the impact of different timings and dosages of slow-release nitrogen fertilizer on the yield, biomass accumulation and distribution, and nitrogen absorption and nitrogen utilization in the DSCWH cropping system. This study was investigated at the experimental farm of Yangzhou University, China in 2020 and 2021, with the short-season cotton variety “Zhongmian 50” used as experimental material. Three dosages of the slow-release nitrogen fertilizer (45 kg·ha−1, 90 kg·ha−1, and 135 kg·ha−1) were applied at two stages of growth (two-leaf and four-leaf). The results showed that applying a 90 kg·ha−1 dosage at the two-leaf stage achieved the highest yield, which was increased by 12.6% compared to the no-fertilization control. Applying 90 kg·ha−1 of the slow-release nitrogen at the two-leaf stage promoted biomass accumulation, especially in reproductive organs, and this increase in biomass of reproductive organs was attributed to optimum nitrogen accumulation in reproductive organs (80~140 kg·ha−1). In addition, when 90 kg·ha−1 was applied at the two-leaf stage, there was a significant enhancement in nitrogen recovery efficiency (NRE), nitrogen agronomic use efficiency (NAE), and nitrogen physiological efficiency (NPE), with increases of 7.2% to 13.0%, 5.7% to 5.8%, and 5.6% to 6.5%, respectively. These results revealed that applying slow-release nitrogen fertilizer with the optimal dosage at the seedling stage could significantly enhance nitrogen use efficiency, nitrogen accumulation and partitioning, and biomass accumulation and distribution, which ultimately resulted in a higher lint yield in DSCWH. Therefore, to optimize yield and NUE, 90 kg·ha−1 slow-release nitrogen applied at the two-leaf stage would be recommended in the direct-sown cotton after wheat harvest cropping system.