It is well known that microcystins (MCs) are the most abundant toxins produced by cyanobacteria in freshwater. The separation and characterization of MCs isomers are very important to the research of algal pollution in freshwater. In this paper MCs isomers were extracted by using methanol water solution and separated by reverse phase high performance liquid chromatography (HPLC). The different isomers were characterized by using HPLC-MS method. Different ratio of extract solvent and chromatographic conditions were discussed. Five MCs isomers were successfully extracted from cyanobacteria of Dianchi Lake. Three of which were characterized to be MC-RR, MC-YR and MC-LR, 1.5mg (92.3 purity) of MC-LR was prepared by using a semi-preparation HPLC system.