An increase in free radicals in the body causes oxidative stress. Therefore, our body needs exogenous antioxidants. This study aimed to determine the antioxidant activity of crude and defatted polysaccharide extracts from water lettuce (Pistia stratiotes) leaf. The polysaccharide was extracted by hot-water extraction and defatted with acetone. The total sugar content was determined by the Phenol-Sulfuric acid method, the antioxidant activity by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method, and the functional group was analyzed by Fourier transform infrared (FT-IR) spectroscopy. The results showed that the total sugar of the crude polysaccharide (96.80±14.60 mg glucose eq./g dry sample) had a higher total sugar than the defatted polysaccharide (44.0±2.16 mg glucose eq./g dry sample). However, the antioxidant activity in defatted polysaccharides (IC50 0.406±0.011 mg/mL) more effectively inhibits free radicals than crude extract (IC50 0.484±0.028 mg/mL). The O-H, C-H, C=O, and C-O functional groups were identified in the polysaccharide extracts. The polysaccharides extract from water lettuce exhibits antioxidant activity. Therefore, the polysaccharide from water lettuce (P. stratiotes) leaf can be used as a source of antioxidant agents and as a food supplement.