Agglutinins are carbohydrates applied as tools in cell biology, immunology, in the study and treatment of cancers, and in a range of areas of biochemical and biomedical research. This study aims to determine the best method of possible hemagglutinating substances extraction from Chlorella vulgaris biomass using three different buffers. The cultured was concentrated by a centrifugation at 4,000 xg for 10 min, lyophilized and extracted under homogenization in pure and in concentrations of 1:5 to 1:20 using the buffers 0.025M Tris-HCl-saline, pH 7.0; 0.02M saline-phosphate, pH 7.4; and 0.1M sodium acetate, pH, 5.5. In microtiter boards, biomass extracts were added to rabbit erythrocytes for hemagglutination activity. Positive results were obtained in Tris-HCl-saline buffer (1:10 and 1:20 dilutions) and sodium acetate buffer (pure extract and dilutions) with hemagglutination titers of 212 (4,096) at 3h of homogenization. Microalgae are promising sources of useful bioactive compounds.