Tofu whey is a yellow drain produced during the production of soy products. It contains a large amount of nutrients that may cause waste of resources and environmental pollution if discharge it directly. Therefore， it is of great significance to comprehensively develop and utilize tofu whey. In this study， we use five Lactobacillus species （Lactobacillus harbinensis M1， Lactobacillus mucosae M2， Lactobacillus fermentum M4， Lactobacillus rhamnosus C1， Lactobacillus amylolyticus L6）， which were isolated and screened from natural acidified tofu whey， and use Streptococcus thermophilus ST3， which was isolated and screened in the commercial starter， to ferment tofu whey. The changes of metabolites and volatile components before and after fermentation were investigated by nuclear magnetic resonance （NMR） spectroscopy and gas chromatography-mass spectrometry（GC-MS）. The results showed that 25 substances were detected in the NMR spectrum， including sugars， acids， amino acids， biologically active 4-aminobutyric acid （GABA） and choline； 95 kinds of volatile flavor components were detected by GC-MS from fermented tofu whey. Most of the aldehydes disappeared after fermentation. The ester and ketone content of C1+ST3 group and L6+ST3 group increased respectively. Short chain fatty acids acetic acid， propionic acid and butyric acid were detected by both analytical methods. This result indicates that the metabolism of Lactobacillus produced many functional compounds and flavor components in tofu whey， which laid the foundation for the development and application of tofu whey.