The influence of riboflavin (B-2)-overproducing lactobacilli on the antioxidant status, isoflavone conversion, off-flavor reduction, amino acid profile, and viscosity of B-2-bio-enriched fermented soymilk was investigated. Results showed that B-2 in fermented soymilk was notably increased from 0.2 to 3.8 mu g/mL for Lactobacillus fermentum UFG169 and to 1.9 mu g/mL for Lactobacillus plantarum UFG10. The apparent viscosity significantly changed with rising acidity and agglutination of protein. The off-flavor volatile substances (hexanal and nonanal) were significantly reduced in fermented soymilk. Furthermore, a large amount of glucoside form isoflavones was deglycosylated into bioactive aglycones after 4 h up to 32 h. B-2 content and isoflavones significantly improved the antioxidant status of soymilk. Partial least squares regression analysis correlated the strain activity and fermentation time with the improved nutritional and functional soymilk qualities. This study demonstrated the strategy for strain development for B-2-bio-enriched fermentation to extend the health-promoting benefits of soymilk and soy-related foods.