SummaryFrom recent literature and research studies, it has been found that some bacterial strains have abilities with probiotic activities to produce B vitamins, which play essential roles in maintaining human health. Oligosaccharides can promote the beneficial microbes, however, the specific effects of oligosaccharides on B vitamins‐producing ability of probiotic strains have not been clarified. This study evaluated the effect of different oligosaccharides on the probiotic properties and B vitamin‐producing ability of 21 selected probiotic strains. The obtained results suggested that the strains could utilise oligosaccharides as the sole carbon source, and the hydrophobicity, auto‐aggregation, and biofilm formation of probiotic strains were specific regulated after fermentation with different oligosaccharides. In particular, the oligosaccharides had different influences on the levels of extracellular B vitamins produced by probiotic strains. These findings provide the foundation for studies on the B vitamins‐producing probiotic strains and their application in functional foods to prevent B vitamins deficiency.