Objective: To present a microbiological method for the detection of biotin, folic acid and vitamin B12 in infant formula powder. Methods: For quantification of these vitamins, the growth of the lactic acid bacteria with specific sensitivity to them was measured after incubation in their presence. Thirteen samples of commercial infant formula powder were detected by the proposed method. Results: More stable and repeatable data were obtained from the determination of biotin and vitamin B12 by the microbiological method with a relative standard deviation (RSD) of 0.056%～4.158% and 0.112%～3.848%, respectively, while the results obtained for folic acid varied in a wide range with a RSD of 1.236%～8.332%. Conclusion: This method is suitable for the quantitative analysis of the vitamins biotin, folic acid and vitamin B12 in infant formula powder.