Natto is a famous traditional fermented food, but the influence of the fermentation process on the content and composition of soybean isoflavones and nutritional value is still unclear. In the present study, the variation in soybean isoflavones during fermentation by Bacillus subtilis natto was revealed by UPLC-ESI-MS/MS (Ultra high performance liquid chromatography-electron spray ionization-mass spectrometry) analysis. After 24 h of fermentation, the total isoflavone content in natto increased by 1.62 times compared with fresh soybean, and the content of aglycones was 3.07 times that of raw beans. More importantly, among 14 isoflavone isomers identified in natto, the isomers of daidzin, genistin, and succinyl genistin were detected for the first time, which might be due to the result of isomerase and succinylase and other corresponding enzymes’ action in Bacillus subtilis. In addition, natto isoflavones performed great antioxidant activity than its monomer components (glycosides daidzin and genistin, aglycones genistein and daidzein), except for genistein. Moreover, natto isoflavone and its aglycones (especially genistein) performed great inhibitory activity against AGEs (Advanced Glycation End Products) in three in vitro models. The mechanism test showed that genistein could form adducts (UPLC-Q-TOF-ESI-MS/MS analysis) with methylglyoxal. These findings demonstrated that soybean fermented with Bacillus subtilis natto had a significant influence on the isoflavone profiles and its bioactivity.