Understanding the response of different alfalfa varieties to nitrogen fertilizer application for nodulation and nitrogenfixation，and enhancing the synergy between alfalfa nitrogen fixation ability and yield andquality，are crucial for theproduction practice of alfalfa nitrogen conservation and efficiency enhancement. In thisstudy，ten alfalfa varieties were usedand five nitrogen gradients were set up. By measuring the abovegroundbiomass，crude proteincontent，root nodulenumber，root nodule weight，nitrogen rate and amount fixed from the atmosphere，the effects of nitrogen application on thebiomass，crude protein content，and nodule nitrogen fixation of different alfalfa varieties in the year of establishment and their mutual regulatory relationship were explored. The results indicated that there were significant differences in aboveground biomass and crude protein content among different varieties and treatments. The nitrogen fixation efficiency of varieties with higheraboveground biomass and crude proteincontent，such asChaoxinxing，Baimu202，QishiT，Baimu 341 andLvsile，wassignificantly higher than that of other varieties. The response of nitrogen derived from the atmosphere to nitrogen applicationrate showed significant differences between two alfalfa cuts. The first cut had the highest response under the N0 treatment， and the second cut or only one cropping had the highest response under N25 and N50 treatments. The crude protein contentshowed a positive correlation with it. Under N0-N50 treatments，the biomass and crude protein content of high nitrogen fixation efficiency varieties were 1.2%-16.7% and 12.9%-19.2% higher than those of low nitrogen fixation efficiencyvarieties，respectively. Compared with N100 and N150treatments，except for the first cut with lower nitrogen derived fromthe atmosphere，the biomass difference was basically the same，and the crude protein content difference was significantly reduced to 7.8%-12.3%. The crude protein accumulation of low nitrogen fixation efficiency varieties was more dependent on nitrogen application. There was a significant positive correlation between biomass and crude protein content and nodulationnitrogen fixation indicators. In the year ofplanting，appropriate nitrogen application was beneficial for the coordinated andefficient nitrogen fixation of alfalfa. Five varieties with higher nitrogen fixationefficiency，such asChaoxinxing，couldachieve a balanced increase in biomass and crude protein content under lower nitrogengradients，and were significantly higher than other varieties. They could be used as excellent varieties for nitrogen-saving cultivation.