Cover cropping combined with nitrogen (N) application reduction is considered as an attractive practice due to increasing resources use efficiency while improving soil health and cash crop productivity. Yet comprehensive assessment studies have rarely been reported from energy, environmental, economic and ecological perspectives under different cover cropping systems. Therefore, a field experiment was conducted during 2019–2022 with four N application rates (i.e., 0 (N0), 112.5 (N1), 168.75 (N2) and 225 (N3, as a control) kg N hm−2) under four cover cropping systems (i.e., continuous cotton (CC), Orychophragmus violaceus with cotton (OC), Vicia villosa with cotton (VC), and an Orychophragmus violaceus and Vicia villosa mixture with cotton (MC) relay intercropping). Field observations were incorporated into a life cycle assessment to evaluate the environmental footprint, energy flow, ecosystem service value and economic and ecosystem economic benefits. Relay intercropping cover crops can reduce the carbon footprint per unit area (CFa) by 36.7%, increase the ecosystem service value by 64.4% and ecosystem economic benefits by 7.8% compared with the CC system, in which MC system had better performance than OC and VC systems at above aspects. N application rates notably increased net energy, CFa, NFa, ecosystem service value, and ecosystem economic benefits. After considering the comprehensive index of energy, environment, the economy and the ecosystem economy, the sustainability performance index of MCN2 (Orychophragmus violaceus and Vicia villosa mixture/cotton relay intercropping with 168.75 kg N hm−2) combination increase by 51.7% compared to all treatments average. Taken together, the study results demonstrated that introducing cover crop mixture into monoculture cotton was more sustainable and cleaner cotton production practice than sing cover crop or fallow in the Yellow River basin and other regions with similar ecological conditions.