Lactic acid bacteria exopolysaccharides (LAB EPS) have garnered significant interest owing to their beneficial biological properties and safety. This study aimed to screen LAB EPS with comprehensive biological activities and anti-allergic potential from four high-yield strains, isolate and characterize their homogeneous polysaccharide fractions, and preliminarily explore their anti-allergic mechanism. Through in vitro evaluation of antibacterial, hyaluronidase inhibitory, antioxidant and immunomodulatory activities, Lacticaseibacillus casei LZ9183 EPS (183 EPS) was identified as the optimal candidate with the most superior comprehensive performance. Further purification via DEAE-52 anion exchange chromatography and Sephadex G-100 gel filtration chromatography yielded two homogeneous polysaccharide fractions, LZEPS-1 and LZEPS-2. Structural characterization results showed that LZEPS-1 (28.73 kDa) was a neutral polysaccharide composed of rhamnose, galactose, glucose and mannose with a total sugar content of 94.39 ± 0.92%, while LZEPS-2 (95.58 kDa) was an acidic polysaccharide containing glucose, galactose, arabinose, mannose, xylose and glucuronic acid with 92.17 ± 1.22% total sugars and 1.72 ± 0.04% protein. Using the RBL-2H3 cell degranulation model, we demonstrated that 183 EPS, LZEPS-1 and LZEPS-2 effectively alleviated IgE-mediated allergic responses by inhibiting the release of β-hexosaminidase and histamine, and modulating the secretion of IL-4 and TNF-α to restore the Th1/Th2 balance. These results propose that LAB EPS could potentially be utilized as natural anti-allergic functional ingredients in the development of functional foods or dietary supplements.