Core Ideas Defoliants enhanced seed cotton yield by increasing opening boll number. N application promoted cotton yield by increasing seed cotton weight per boll. 150 kg N ha−1 and thidiazuron+ethephon of 900+3000 g ai ha−1 was suggested. Appropriate nitrogen levels combined with safe rates of harvest aids can provide timely and efficient harvesting of cotton in the wheat–cotton double cropping system in the Yangtze River Valley Cotton Belt. Three rates of harvest aids (0+0, 450+1500, and 900+3000 g ai ha−1 of thidiazuron+ethephon) under four nitrogen levels (0, 75, 150, and 225 kg N ha−1) were established to study the interactive effects of N levels and harvest‐aid rates on defoliation, boll opening, and seed cotton yield in 2015 and 2016. Results showed that defoliation and boll opening percentages were significantly enhanced by 45 to 57% and by 35 to 41% after the application of harvest aids, especially for the elevated rate, under 150 and 225 kg N ha−1. Although a significant negative effect was observed on seed cotton weight per boll, total seed cotton yield was significantly increased by 28 to 42%, due to the favorable opening boll number after harvest‐aid application. Nitrogen application significantly increased seed cotton yield by 10 to 22%, owing to the enhanced seed cotton weight per boll, and the maximum total seed cotton yield was recorded under 150 kg N ha−1 when applied with harvest‐aid chemicals of 900+3000 g ai ha−1. Thus, the application of 150 kg N ha−1 combined with 900+3000 g ai ha−1 of harvest aids (thidiazuron+ethephon) was suggested for mechanized harvest in cotton to achieve a satisfactory defoliation and seed cotton yield in the Yangtze River Valley Cotton Belt.