Abstract: Objective During the processing of sea cucumbers, large amounts of cooking liquid are generated, which not only contributes to environmental pollution but also results in the loss of valuable nutrients. This study aimed to extract polysaccharides from sea cucumber cooking liquid, analyze their physicochemical properties and antioxidant activity, and establish a scientific foundation for the utilization of these polysaccharides. Methods Polysaccharides were extracted using enzymatic hydrolysis combined with cetylpyridinium chloride precipitation. The basic components of the polysaccharides was determined using the Kjeldahl method, phenol-sulfuric acid method, high-temperature ashing method, and barium chloride precipitation method. Structural characteristics were analyzed via high-performance liquid chromatography, Fourier-transform infrared spectroscopy, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). In vitro antioxidant activity was evaluated by measuring DPPH radical, ABTS radical, and hydroxyl radical scavenging capacities. Results The polysaccharides contained 34.92% ± 0.16% total sugar, 16.31% ± 0.04% protein, 10.49% ± 0.11% ash, and 22.85% ± 0.09% sulfate. The monosaccharide composition (mass percentage) was determined as Man∶GlcN∶GlcUA∶GalUA∶GalN∶Gal∶Fuc=36.55∶6.00∶7.39∶8.76∶11.02∶8.45∶23.83. DSC analysis revealed an endothermic peak at 176.34°C (enthalpy: 114.69 J/g) and an exothermic peak at 232.32°C (enthalpy: 108.49 J/g). SEM images showed a loose honeycomb-like structure at 200× magnification and intact surfaces with protrusions at 5,000× magnification. At 4 mg/mL, the DPPH radical scavenging rate reached 93.97%, while ABTS and hydroxyl radical scavenging rates at 2 mg/mL were 91.12% and 65.49%, respectively. The IC₅₀ values for DPPH, ABTS, and hydroxyl radical scavenging were 1.65, 2.09, and 2.12 mg/mL, respectively. Conclusion Sea cucumber cooking liquid polysaccharides are polysaccharides composed of seven monosaccharides: mannose, glucosamine, glucuronic acid, galacturonic acid, glucosamine, galactose, and fucose. Among them, mannose has the highest proportion, followed by fucose. The polysaccharides exhibit strong thermal stability and significant antioxidant activity in vitro, demonstrating promising potential for development as functional food ingredients.