Rational intercropping is capable of promoting nodulation and facilitating the fixation and utilization of nitrogen (N) of legumes. Isoflavonoid is critical to form root nodules of legumes, whereas the regulation of isoflavonoid over nodulation and N fixation in legume/cereal intercropping remains unclear. In the present study, nutrient solution of different root partitions (e.g., no barrier (A-O), nylon mesh barrier (NA-O), plastic barrier (PA-O) and sole alfalfa (SA)) and nitrogen levels (e.g., 21 and 210 mg L-1 N) were used to delve into the variations of nodulation, N fixation, isoflavonoid content, isoflavonoid synthase (IFS) and nodulation-signaling pathway (NOD) genes in alfalfa. As suggested from the results, the parameters of nodulation and N fixation ability with A-O were all evidently higher than those with PA-O and SA. Formononetin and genistein with A-O had noticeably higher contents than those with PAT and SA. Daidzein and luteolin with A-O had remarkably higher contents than those with NA-O in the root. IFR 1, IFR 4, NOD 1 and NOD 2 with A-O and NA-O had noticeably higher relative expressions than those with SA and PA-O. IFR 2 and IFR 3 with AT and NA-T had significantly lower relative expressions than those with PA-O and SA. The nodulation and isoflavonoids met significant positive associations. IFR and NOD genes and TNN and ENN followed significant positive associations. NOD genes and formononetin exhibited extremely significant positive associations. The conclusion was drawn that the closer root interaction between alfalfa and oat, the more effective the nodulation, N fixation ability and isoflavoids content of alfalfa will be. Isoflavonoids primarily affected nodulation and N fixation. The mechanism of isoflavonoids on nodulation and N fixation in alfalfa/oat intercropping was explained that root interaction results in the reduction of environmental N, thereby regulating the relative expressions of IFR genes, elevating the isoflavonoids contents, and up-regulating the relative expressions of NOD genes; as a result, the nodulation and N fixation ability in alfalfa are facilitated.