Abstract: Using polysaccharide extraction yield as the indicator, this study investigated the effects of hot water extraction (HWE), enzyme-assisted extraction (EAE), and semi-bionic extraction (SBE) on the extraction yield of Prunella vulgaris L. polysaccharides. The extraction method with the highest polysaccharide yield was selected for process optimization. The structure of the polysaccharides was characterized using high-performance liquid chromatography, gel permeation chromatography, ultraviolet spectroscopy, and Fourier-transform infrared spectroscopy. In addition, the in vitro antioxidant and enzyme inhibitory activities were evaluated. The results showed that the polysaccharide yields obtained by the HWE, EAE, and SBE were 5.9%, 7.54%, and 9.87%, respectively. After optimization, the best extraction conditions for the SBE method were determined as follows: extraction temperature of 85 °C, duration of 2.5 h, simulated gastric fluid (pH2.0) and intestinal fluid (pH8.5), and a solid-to-liquid ratio of 1:30. Under these conditions, the polysaccharide yield reached 11.69%. Structural analysis revealed that Prunella vulgaris polysaccharides were mainly composed of rhamnose, galacturonic acid, glucose, galactose, xylose, and arabinose, the molar ratios were 8.89, 41.81, 17.53, 6.85, 14.32 and 7.21, respectively. Antioxidant activity tests showed that the IC₅₀ values for scavenging DPPH·, ABTS⁺·, ·OH and O₂⁻· were 0.049, 0.092, 0.085 and 0.062 mg/mL, respectively, while the maximum total reducing power was measured at 0.314 when the polysaccharide concentration was 1.1 mg/mL. The polysaccharide also exhibited inhibitory effects on α-glucosidase and α-amylase, with IC₅₀ values of 1.72 and 8.43 mg/mL, respectively. This study systematically optimized the extraction process of P. vulgaris L. polysaccharides, preliminarily clarified their structural characteristics, and confirmed their strong antioxidant activity. These findings provide theoretical basis and technical support for the further development and application of P. vulgaris L. polysaccharides.