The polysaccharide was from Spirulina powder by the means of hot water extraction after repeated freezing-thawing cycles and ultrasonic treatment. The effects of lasted time and extraction temperature and time, ratio of solid to liquid and times of extraction on polysaccharide yield were studied by single factor and orthogonal experiments. PSP was separated into PSP-3 by membrane separation techniques, and it was investigated by MTT assay on colon adenocarcinoma cells (Caco-2 cell growth inhibition) to determine the optimal concentration. Finally, the molecular of PSP-3 was determined by gel permeation chromatography (GPC). Experiment results showed that the optimum conditions were as follows: ultrasonic treating time 20 min, extracting temperature 90 ℃, ratio of material to liquid 125, extracting time 1.5 hours, extracting time twice. Under the conditions, the yield reached (13.17 ± 0.49)%, being 1.59 times of the polysaccharides prepared by traditional water extraction. PSP was separated and purified into PSP-3 and its inhibition on colon adenocarcinoma cells (Caco-2 cells) was investigated. The results indicated that the molecular weight of PSP-3 was 623.02 kDa and the optimum inhibitory concentration was 5.00 mg/mL, with IC₅₀ value of 0. 63 mg/mL.