OBJECTIVE To optimize the deproteinization process of Isatidis Radix polysaccharides and further explore its immu-nomodulatory activity,and to provide a scientific basis for the development and utilization of it.METHODS The optimum conditions of enzymatic deproteinization were optimized by a single factor combined with the Box-Behnken response surface method.The chemical composition and structural characteristics of deproteinized Isatidis Radix polysaccharides were analyzed by UV-visible spectrum,Fourier transform-infrared spectroscopy,high-performance gel permeation chromatography,high-performance liquid chromatography and scan-ning electron microscopy.The effects of deproteinized Isatidis Radix Polysaccharide on neutrophils,macrophages,IL-1β and IL-6 in zebrafish were investigated by using a zebrafish immunocompromised model.RESULTS The optimal enzymatic deproteinization process was as follows:trypsin 500 U·mL-1,pH 8.0,enzymatic hydrolysis time 5 h,enzymatic hydrolysis temperature 37℃.The deproteinization rate was(86.39±0.07)%,and the comprehensive score was(91.15±0.37)%.Ultraviolet,infrared spectroscopy scanning and scanning electron microscopy showed that the protein contained in the crude polysaccharide could be removed by enzymat-ic method.The relative molecular weight of the polysaccharides were between 5.82 and 60.26 kDa.The monosaccharide mole compo-sition was mannose ∶ rhamnose ∶ galacturonic acid ∶ glucose ∶ galactose ∶ arabinose=2.17 ∶ 0.96 ∶ 2.90 ∶ 83.25 ∶ 4.88 ∶ 5.84.The results of immune activity evaluation showed that when the concentration of deproteinized Radix Isatidis polysaccharides was 50～300 μg·mL-1,it could significantly increase the density of zebrafish immune cells,increase the number of macrophages,and reduce the content of IL-1β and IL-6 in immunocompromised zebrafish,thus exerting immunomodulatory effects.CONCLUAION The enzy-matic method can effectively remove the proteins contained in the crude polysaccharides of Isatidis Radix,and the deproteinized Isatidis Radix polysaccharides have certain immunomodulatory effects.