In order to increase the number of organisms per unit volume of fermentation broth and provide a basis for the development and application of Lactobacillus rhamnosus ZB1107-01 (LR-ZB1107-01) obtained in the early stage of the study, which is well tolerated by simulated gastrointestinal tract, this paper initially performs medium optimization and probiotic properties investigation of the strain. First, the growth curve of strain LR-ZB1107-01 in MRS medium was measured to clarify its growth pattern in liquid medium. Next, the number of viable bacteria in the fermentation broth of strain LR-ZB1107-01 was used as the detection index, and different kinds of nitrogen sources, carbon sources and growth factors were examined unilaterally to determine the optimal medium components, and response surface tests were conducted to optimize the medium ratios and select the best medium. Finally, the in vitro safety of the nitro reductase activity and amino decarboxylase activity was evaluated. The results show that the viable bacteria count concentration can reach 8.56×10⁸ CFU/mL, if three nutrients and contents that significantly affected the viable bacteria count are changed as follows: the addition of D-isoascorbic acid at 0.02%, magnesium sulfate at 0.12% and peptone at 3%, based on the glucose content of MRS medium at 2% and combined with the optimized culture conditions. The viable bacteria count is about 10.7 times higher than that in MRS medium. The in vitro safety evaluation results indicate that LR-ZB1107-01 is negative for both nitroreductase and amino acid decarboxylase, suggesting that the strain has a certain safety profile. This achievement has a certain guiding effect on the industrial production of probiotic strains and the subsequent development of products.