The main functional ingredients of hot water extract of Chlorella pyrenoidosa (CPE) were investigated through a bioassay-guided fractionation based on free radical scavenging and macrophage proliferation effects. The main functional ingredients of CPE were polysaccharides (PS) that were isolated by high pressure extraction, Sevag method, ethanol precipitation and ultrafiltration separation. Crude polysaccharides were further separated and purified by ion exchange chromatography DEAE52 and size exclusion chromatography Sephadex G-100. The purified fractions were analyzed by gel permeation chromatography. Molecular weights of the purified fractions PS-1-4-2, PS-1-3-2 and PS-2-3-3 were 3.97×104, 2.28×104 and 4.1×103 Dalton, respectively. Bioassay-guided fractionation results indicated that CPE could remove free radicals and promote Ana-1 cells proliferation, mainly due to its various components working together. The components of free radicals scavenging mainly concentrated in PS-1-3, PS-1-4, PS-2-3 and PS-2-4. The components of Ana-1 proliferation mainly concentrated in PS-1-3, PS-1-4 and PS-2-3. This study established the activity screening method of main functional component from CPE, and got three new functional ingredients. It can be used to guide the development of high value products, further promote the industrialization process of microalgae energy, and realize microalgae ‘high value products, microalgae energy and microalgae carbon’ integration of exemplary role.