Abstract: Selenium-enriched (Se-enriched) lactic acid bacteria (LAB) can convert inorganic Se into organic one to produce new products. In this paper, Se-resistant LAB were screened out from 15 LAB by adding Se with a mass concentration of 20 μg/mL to MRS medium and detecting the OD value. The appropriate Se adding time of Se-enriched LAB was determined by growth curve, and concentration was determined by cell density with different Se contents in the culture medium. The Se accumulation amount of LAB was measured through 3, 3’-diaminobenzidine (3, 3’-DAB) method. The in vitro activity of Se-enriched LAB and its survival ability in human intestinal tract were evaluated by detecting the antioxidant activity, bile salt resistance and artificial gastric fluid resistance of bacteria. Results showed that 9 Se-enriched LAB were screened out from 15, the appropriate addition time was 2 h, concentration was 40 μg/mL, and the strain JX5 with the highest Se conversion rate was 81.80%. 9 strains had different scavenging effects on free radicals DPPH and ABTS after Se enrichment, and had different abilities to resist bile salts and artificial gastric fluid, and the overall in vitro activity showed an increasing trend, especially strain JX5, increased significantly in four indexes, which indicated that the transformation of inorganic Se into organic one by LAB can improve the in vitro activity of bacteria. The antioxidant capacity and bile salt tolerance of 9 strains were improved to some extent.