To　promote　the　high-value　utilization　of　Nannochloropsis　polysaccharides,　gradient　ethanol　precipitation　was　applied　to　separate　Nannochloropsis　soluble　polysaccharide　(NSP).　Three　fractions　(NSP40,　NSP60,　and　NSP80)　were　obtained　by　40%,　60%,　and　80%　ethanol　precipitation　sequentially.　The　physicochemical　characteristics　including　monosaccharide　composition,　molecular　weight,　structure,　and　rheological　properties　of　polysaccharides　were　analyzed　by　HPLC,　high　pressure　gel　chromatography-muitiple　angle　light　scattering　detector-differential　refractive　index　detector,　Fourier　transform　infrared　spectrometer,　and　rheometer.　The　antioxidant　activities　of　each　fraction　were　also　evaluated.　Results　showed　that　the　yield　and　total　sugar　content　of　polysaccharide　precipitated　were　positively　correlated　with　ethanol　concentration.　When　the　ethanol　concentration　was　80%,　the　yield　and　total　sugar　content　was　(3.48　±　0.04)　%　and　(74.43　±　0.76)%,　respectively.　The　content　of　uronic　acid,　monosaccharide　composition,　molecular　weight　distribution,　and　viscosity　of　NSP　were　affected　by　ethanol　concentration.　In　which,　NSP60　possessed　the　highest　average　molecular　weight　and　viscosity.　All　of　the　three　NSP　fractions　isolated　were　heteropolysaccharides　and　composed　of　9　monosaccharides　including　Man,　Gal,　Rib,　Rha,　Fuc,　Xyl,　Ara,　and　GLC-UA.　The　monosaccharide　molar　ratio　of　each　fraction　was　significantly　different.　Antioxidant　analysis　showed　that　NSP40　had　the　strongest　ability　to　scavenge　ABTS　cationic　radicals　and　hydroxyl　radicals,　while　NSP60　had　the　strongest　total　reducing　power.　Therefore,　gradient　alcohol　precipitation　can　apply　to　preliminary　separate　and　obtain　polysaccharide　fractions　with　different　physicochemical　characteristics　and　biological　activities.　©　2025　China　National　Research　Institute　of　Food　and　Fermentation　Industries　Co.　Ltd.　All　rights　reserved.