Based on the DPPH radical scavenging ability, this study employed the Chou-Talalay method to calculate the combination index for analyzing the synergistic antioxidant effect of mixied crude polysaccharides prepared from corn silk polysaccharide (CSP), Hericium erinaceus polysaccharide (HEP), and Ganoderma lucidum polysaccharide (GLP). The content of polysaccharide and protein in mixied crude polysaccharides was determined using the methods of phenol-sulfuric acid and dumas combustion respectively. HPLC and infrared spectroscopy were used to analyze the relative molecular weight, monosaccharide composition, and structural characteristics of the mixied crude polysaccharide. Furthermore, their in vitro antioxidant activities were evaluated, and their in vivo antioxidant effects were investigated using a zebrafish embryo oxidative stress model. The results indicated that HEP and CSP exhibited synergistic effects at mass ratios of 1∶9, 2∶8, and 3∶7. When GLP was added in varying proportions to the HCSP-30 which was obtained by 3∶7 , TCSP-15 ( GLP accounted for 15%) demonstrated the optimal synergistic effects. TCSP-15 exhibited significant antioxidant activity, with IC₅₀ values for scavenging DPPH radicals, hydroxyl radicals, and ABTS⁺ radicals of 138.73 ± 2.43 μg/mL, 1.79 ± 0.01 mg/mL, and 33.70 ± 0.24 μg/mL respectively. The polysaccharide content of TCSP-15 was 47.08% ± 0.29%, while the protein content was 17.96% ± 0.16%, the molecular weight of the main polysaccharide was approximately 1.39×10⁴ Da. The main monosaccharides were mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose, with glucose and galactose being the most abundant. TCSP-15 exhibited typical β-configuration and pyranose characteristics. In in vivo experiments, TCSP-15 effectively reduced zebrafish embryo mortality, improved developmental outcomes, mitigated oxidative stress levels, and enhanced antioxidant enzyme activity. These findings indicate that TCSP-15 provides protective effects against oxidative stress in zebrafish oxidative stress models.