[Objective] Nitrogen and phosphorus loss characteristics and soil fertility changes on a gentle slope of cultivated land were analyzed under optimal fertilization and cross-slope tillage in order to provide a basis for reducing nitrogen and phosphorus losses, achieving non-point source prevention and treatment, protecting cultivated land resources, and maintaining soil fertility level. [Methods] The study was conducted on a gentle slope area of the mountain and hilly area in Yunnan Province under natural rainfall conditions. A surface runoff monitoring area was established with three treatments: down-slope tillage+conventional fertilization; cross-slope tillage+conventional fertilization; and cross-slope tillage+optimal fertilization. The characteristics of nitrogen and phosphorus loss under different tillage and fertilization methods were continuously monitored from January 2019 to December 2021, and soil fertility level was analyzed by the gray correlation method. [Results] There was a significant linear correlation between nitrogen and phosphorus losses and rainfall and runoff (p＜0.01). Compared with down-slope tillage, cross-slope tillage reduced surface runoff by 20.46%—40.74%, total nitrogen loss by 19.12%—33.71%, and total phosphorus loss by 33.33%—60.08%. Compared with conventional fertilization, optimized fertilization reduced total nitrogen loss by 1.20%—51.71%. All soil fertility factors showed different degrees of improvement except that the contents of nitrate nitrogen and organic matter decreased. The gray correlation degree of each treatment showed different degrees of improvement, and the gray correlation degree of the cross-slope tillage+optimized fertilization treatment increased significantly (p＜0.05), with an increase of 26.63%. [Conclusion] Cross-slope tillage stopped surface runoff and reduced the loss of nitrogen and phosphorus. The application of cross-slope tillage and optimal fertilization application had the greatest effect on improving soil fertility level and reducing nitrogen and phosphorus losses.