Objective To clarify the main components of Lycium barbarum polysaccharides that exert an anti-fatigue effect.Methods Lycium barbarum polysaccharides with different relative molecular weights are prepared by water extraction, ultrafiltration membrane fractionation dialysis, and other methods. The components with good anti-fatigue activity are obtained by mice burden swimming test. They are then eluted and purified by ion exchange column and gel chromatography column to determine the relative molecular weights and monosaccharide components. The infrared spectrum is employed to analyze.Results The content of polysaccharide, protein, and uronic acid in Lycium barbarum polysaccharides with different molecular weights is 63.62%~75.63%, 1.77%~2.63%, and 17.77%~24.77%, respectively. The LBP2 has a higher component yield. The relative molecular weight of the four components is 115.3×10³, 47.4×10³, 8.14×10³, and 4.82×10³, respectively. The LBP2 is superior to other components in prolonging swimming time, reducing urea nitrogen and blood lactate content, increasing muscle glycogen and liver glycogen content, and enhancing the activity of superoxide dismutase, peroxidase, and glutathione peroxidase (P<0.05); LBP2 is purified to obtain acidic polysaccharides LBPE1, LBPE2, and LBPE3. The relative molecular weight of LBPE1 and LBPE2 is 3.54×10⁴ and 1.10×10⁴, respectively. The main components are mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose, with molar ratios of 0.73∶1.61∶0.53∶3.22∶6.33∶8.26 and 1.94∶1.81∶1.36∶6.77∶8.46∶6.57, respectively. LBPE1 and LBPE2 have multiple distinct polysaccharide characteristic absorption peaks, both of which are β-pyranoside polysaccharides.Conclusion The polysaccharides LBPE1 and LBPE2 from Lycium barbarum polysaccharide are the main structural components that exert an anti-fatigue effect.