The effect of different fermentation temperature， fermentation time， inoculum amount and pre-culture time on acid fermentation of fermented tofu whey was studied by using a strain of Lactobacillus rhamnosus， which was bred by mutagenesis in the early stage of the research group， using the fermented tofu whey as the medium， acid production and pH value as the index. On the basis of single factor experiment results， orthogonal experiment was used to optimize fermentation conditions， analyze acid production characteristics of the strain， and discuss its metabolic pathway. The results showed that the acid yield of Lactobacillus rhamnosus was 43.11 g/L under the conditions of fermentation temperature 37 ℃， fermentation time 16 h and inoculation amount 5%， which was 9.7% higher than that of the strain before mutagenesis. The contents of lactic acid， citric acid， succinic acid， pyruvate， malic acid， α-ketoglutaric acid and fumaric acid in pure strain fermentation were 1.22， 1.13， 3.14， 1.84， 4.47， 1.18， 49.00 times higher than those in pure strain fermentation without mutagenesis， respectively.