The improvement effect of microbial fermentation on in vitro antioxidant properties of Gracilaria lemaneiforms slurry polysaccharide was studied to provide theoretical support for high value utilization of Gracilaria lemaneiforms. The structure changes of polysaccharide before and after fermentation by Lactobacillus RP38 and Saccharomyces cerevisiae JJ4 were analyzed by modern chromatography and spectral analysis techniques. The free radical scavenging ability of slurry polysaccharides before and after fermentation was investigated. The result showed that the mass fraction of polysaccharide increased from 0.30% to 0.79%, and sulfated polysaccharide mass fraction increased from 0.03% to 0.12%. Results of gel permeation chromatography and infrared spectroscopy revealed that functional groups of Gracilaria lemaneiforms polysaccharides remained unchanged after biological fermentation, the molecular weight of the polysaccharide was reduced, and then more sulfate groups exposed. In addition, it was found by nuclear magnetic resonance scanning that sulfate group of sulfated polysaccharide was mainly distributed in the C-6 position of galactose. The distribution position of sulfate was not changed by biological fermentation, but the content of sulfate was improved. The hydroxyl radical scavenging ability of Gracilaria lemaneiforms polysaccharide was increased by 5.33% and the superoxide radical scavenging ability was increased by 32.12% after fermentation. In conclusion, the antioxidant properties of Gracilaria lemaneiforms slurry could be improved by Lactobacillus RP38 and Saccharomyces cerevisiae JJ4, which may be related to the fact that the formation of small molecular polysaccharide with hydrogen supply capacity and more sulfate groups exposure during fermentation.