Abstract: To screen lactic acid bacteria with high folate production capacity, casein-folate medium was adopted for primary screening, and high-performance liquid chromatography (HPLC) was used to determine folate production in this study. Meanwhile, simulated gastrointestinal fluids tolerance assays, together with measurements of hydrophobicity, auto-aggregation capacity and antioxidant activity, were conducted to systematically evaluate the probiotic properties of strains. Hemolysis tests and antibiotic susceptibility tests were performed to assess their biosafety. Four lactic acid bacterial strains were isolated from traditional fermented foods and all were identified as Lactobacillus plantarum, designated strains 5, 11, 16, and 18. Their total folate yields were (27.03±1.96) μg/mL, (21.21±1.46) μg/mL, (18.02±1.36) μg/mL and (7.49±0.62) μg/mL, respectively. Among the four strains, strain 5 exhibited the optimal tolerance to simulated gastrointestinal fluids, with survival rates of 77.92%±0.68% in gastric fluid and 60.58%±1.71% in intestinal fluid. All strains demonstrated strong hydrophobicity toward ethyl acetate and xylene, ranging from 67.01% to 93.59%. Their auto-aggregation rates ranged between 62.10% and 78.64%. The four strains possessed radical scavenging capacities against DPPH, ABTS⁺, and hydroxyl radicals. The strain 5 achieved high DPPH and ABTS⁺ radical scavenging rates of 82.32%±1.36% and 70.60%±1.14%, respectively, whereas the strain 11 had the highest hydroxyl radical scavenging rate of 60.01%±1.24%. These Lactobacillus plantarum strains possess notable probiotic potential. Furthermore, all four strains were non-hemolytic and sensitive to most antibiotics, indicating their potential for further safety evaluation and preliminary confirmation. This study enriches the resource library of folate-producing lactic acid bacteria and systematically verifies their probiotic properties. To some extent, this addresses the common issues of existing folic acid-producing strains, such as limited functional diversity, an incomplete comprehensive evaluation system, and restricted practical applications. This research provides a theoretical basis for the development of folate-producing probiotics and their applications in the food industry.To screen lactic acid bacteria with high folate production capacity, casein-folate medium was adopted for primary screening, and high-performance liquid chromatography (HPLC) was used to determine folate production. Meanwhile, simulated gastrointestinal fluid tolerance assays, together with measurements of hydrophobicity, self-aggregation capacity and antioxidant activity, were conducted to systematically evaluate the probiotic properties of strains. Hemolysis tests and antibiotic susceptibility tests were performed to assess their biosafety. Four lactic acid bacterial strains were isolated from traditional fermented foods and all were identified as L. plantarum, designated L. plantarum 5, 11, 16, and 18. Their total folate yields of (27.03±1.96) μg/mL, (21.21±1.46) μg/mL, (18.02±1.36) μg/mL and (7.49±0.62) μg/mL, respectively. Among the four strains, strain 5 exhibited the optimal tolerance to simulated gastrointestinal fluids, with survival rates of 77.92%±0.68% in gastric fluid and 60.58%±1.71% in intestinal fluid. All strains demonstrated strong hydrophobicity toward ethyl acetate and xylene, ranging from 67.01% to 93.59%. Their self-aggregation rates varied between 62.10% and 78.64%. The four strains possessed radical scavenging capacities against DPPH, ABTS, and hydroxyl radicals. Strain 5 achieved high DPPH and ABTS scavenging rates of 82.32%±1.36% and 70.60%±1.14%, respectively, whereas strain 11 had the highest hydroxyl radical scavenging rate of 60.01%±1.24%. These L. plantarum strains possess notable probiotic potential. Furthermore, all four strains were non-hemolytic and sensitive to most antibiotics, indicating suitability for further safety evaluation and preliminarily deeming them safe. This study enriches the resource library of folate-producing lactic acid bacteria and systematically verifies their probiotic properties. It overcomes the drawbacks of existing folate-producing strains, including limited functional spectra, incomplete functional evaluation and narrow application scope. This research provides a theoretical basis for the development of folate-producing probiotics and their applications in the food industry.