In this study,pot experiments were conducted to study the effects of nitrogen application levels and root separation patterns on the root morphology and nitrogen fixation ability of alfalfa in the maize/alfalfa intercropping system using random block design and root separation technology,so as to provide a scientific basis for reduction and efficiency enhancement of chemical fertilizer in the maize/alfalfa intercropping system. The results showed that nitrogen application increased the root length,root surface area and root volume of the alfalfa at different diameter-class by 22.4%, 18.4% and 16.9%, but decreased the number of nodules,dry weight of the nodules,the rate of nitrogen fixation,fixed amount and the leghemoglobin content by 33.8%, 64.5%, 29.4%, 17.0% and 55.4% respectively.Under no nitrogen fertilizer(N0) treatments, compared with plastic sheet separation (PSS) treatment, the root length,root surface area and root volume of alfalfa at different diameter-class of no separation(NS) and nylon net separation (NNS)treatments were reduced by 26.1%,29.1%,36.9% and 13.6%,12.9%,23.5%,and the number of root nodules,dry weight of root nodules,nitrogen fixation rate,fixed amount and the content of leghemoglobin of NS and NNS treatments were increased by 69.5%,76.4%,148.2%,52.9%,73.3% and 49.3%,61.4%,66.0%,41.9%,43.3%,respectively;Compared with the NNS treatment,the root length,root surface area and root volume of alfalfa at different diameter-class of NS treatment were reduced by 14.0%,18.8% and 19.5%,and the number of alfalfa nodule,dry nodule weight,nitrogen fixation rate, fixed amount and the content of leghemoglobin of NS treatment were increased by 13.5%,9.3%,49.5%,7.8% and 20.9%respectively.Under nitrogen fertilizer (N1) application treatment,compared with PSS treatment,the root length,root surface area and root volume of alfalfa at different diameter-class of NS and NNS treatments were reduced by 23.9%,28.3%,32.3% and 12.6%,15.3%,23.5%,and the number of root nodules,dry weight of root nodules,nitrogen fixation rate,fixed amount and the content of leghemoglobin of NS and NNS treatments were increased by 45.5%,97.6%,105.5%,30.0%,42.6% and 42.8%,45.5%,58.6%,26.2%,18.1%,respectively;Compared with the NNS treatment,the root length,root surface area and root volume of alfalfa at different diameterclass of NS treatment were reduced by 12.9%,15.4% and 18.1%,and the number of alfalfa nodule,dry nodule weight,nitrogen fixation rate,fixed amount and the content of leghemoglobin of NS treatment were increased by 1.9%,35.8%,29.6%,2.9% and 20.7%,respectively.Therefore,both nitrogen application and root separation promote the growth of root at different diameter-class in the maize/alfalfa intercropping system,while inhibite the nitrogen-fixing ability of alfalfa.