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A sulfated polysaccharide from Navicula incerta (SPNi) was extracted, and its physicochemical characteristics, antioxidant activity, and anti-hemolytic property were investigated. The polysaccharide yield was 4.8% (SPNi weight/biomass dry weight). Glucose, galactose, mannose, and xylose were the primary sugars. The sulfate content and Mw values were 0.46% and 45 kDa, respectively. The FT-IR spectrum showed characteristic bands at 3276,1079,1255, and 820 cm−1, related to -OH, C-O-C, S=O, and C-O-S stretching vibration. The 1H-NMR analysis revealed signals of anomeric protons, indicating the presence of CH2-O and CH-O groups. SPNi registered ferric-reducing antioxidant power (up to 1.47 µmol TE/g) and 54% anti-radical activity on ABTS+. This polysaccharide registered 90% hemolysis inhibition achieving integrity of the erythrocyte membrane. The results indicate that SPNi could be a candidate for biotechnology applications where antioxidant activity and hemolysis inhibition are required.