The study screened folic acid production strains from breast milk， infant feces， dairy products， and sour meat. The production of folic acid was increased through medium optimization and metabolic pathway study. Among 39 folate-producing strains， 4 high-yielding strains were obtained: Lactobacillus rhamnosus B2-14 (39.56 ng/mL)， Bifidobacterium D020 (32.56 ng/mL)， Lactobacillus reuteri B1-28 (60.72 ng/mL)， and Lactobacillus formans F020 (61.22 ng/mL). By optimizing the medium conditions， folic acid production can be increased by 50%-70%. When cultured in milk medium， folic acid yield can be increased by 6-10 times. Among them， the yield of strain B1-28 reached 910 ng/mL after being cultured in milk medium. Some indexes of the folic acid production pathway and related metabolic genes were analyzed to explore changes in the synthesis pathway indexes and their mechanisms for developing dairy products conducive to folic acid intake.