Lactic acid bacteria (LAB) are useful microbes for producing food and pharmaceutical science. To investigate the potency for the production of vitamins by lactic acid bacteria are necessary. In this study, vitamins B6 and B9 produced by Lactobacillus rhamnosus PN04. By high perform liquid chromatography analysis, Lactobacillus rhamnosus PN04 could produce vitamin B6, vitamin B9, equaled to 0.062 mg/ml, 0.002 mg/ml, respectively. The results were also confirmed using thin-layer chromatography analysis. Only vitamin B6 (Pyridoxine) was detected due to vitamin B9 was low. As a result, Lactobacillus rhamnosus PN04 could be the vitamin source for pyridoxine and folic acid.