Abstract: To prepare Eucheuma spinosum sulfated polysaccharides with low viscosity, high solubility and high bioavailability, fermented Eucheuma spinosum sulfate polysaccharides (F-ESP) was prepared by fermenting Lactobacillus rhamnoides, a common probiotic bacteria in fermented food, response surface methodology was used to optimize the preparation process with the yield of crude polysaccharide as the index. Low temperature freeze-thaw Eucheuma spinosum sulfate polysaccharide (L-ESP) was used as control. After purification by DEAE-52 column chromatography, the contents of total sugar, reducing sugar, sulfate and other substances and monosaccharide composition were determined, and the chemical composition of F-ESP was analyzed. The physical properties of F-ESP were characterized by viscosity, solubility, molecular weight and microstructure. The functional group structure of F-ESP was analyzed by infrared spectroscopy. The antiallergic activity of F-ESP was evaluated by Rat Basophilic Leukemia-2H3 (RBL-2H3) degranulation assay. When the ratio of material to liquid was 1:70, the inoculation amount was 5%, and the fermentation time was 24 h, the yield of crude polysaccharide was the highest (41.70%±2.00%). The purified F-ESP-3 monosaccharide structure was mainly composed of galactose, and the total sugar content was 97.77%±1.10%. The sulfate content was 28.40%±1.40%. Compared with L-ESP-3, the reducing sugar content of F-ESP-3 (7.87%±0.09%) was significantly higher, the molecular weight (33.58 kD) was significantly decreased (P<0.01), the specific viscosity (0.01±0.002 dL/g) was significantly lower (P<0.01), and the solubility (89.33%±3.10%) was significantly higher (P<0.0l). Under scanning electron microscope, L-ESP-3 was smooth and dense flake, and F-ESP-3 was rough and irregular granule. The sugar chain structure of F-ESP-3 prepared by fermentation did not change. It was a kind of α-pyranose sulfate containing 3-methylether-galactose residue, and inhibit the activation of basophils by inhibit the release of β-hexosaminidase. To sum up, probiotic fermentation could be used as a way to prepare Eucheuma spinosum sulfated polysaccharides, and its anti-allergic activity could be significantly improved by changing its physical properties. This result provides data support and theoretical basis for the industrial production, development and utilization of Eucheuma spinosum polysaccharides.