Surface coat is a functional structure surrounding cells of cyanobacteria, and the extraction method is still not well compared and consistently reported. Previous work suggested that the main factors affecting the extraction of cyanobacterial surface coat were temperature, extractant concentration, extraction time, etc. To develop the optimal extraction methods for surface coat of cyanobacteria, Anabaena sp. and Nostoc sp. with different morphotypes of surface coat were selected. We performed a series of factors based on two-factor and orthogonal experimental design. Through comparison of cell morphology, spontaneous fluorescence and cell density after surface coat extraction, the results showed that slime can be separated from cyanobacterial cells by centrifugal method. Both sulfuric acid and sodium hydroxide water-bath methods can extract capsule, and the optimal conditions were 1 mol/L, 60℃, 30 min for sulfuric acid extraction and 0.01 mol/L, 50℃, 20 min for sodium hydroxide, respectively. The optimal method of sheath extraction was 0.01 mol/L sodium hydroxide water-bath method, 80℃, 40 min and 20 kHz, 30 W, on:3 s, off:3 s, 2 min by ultrasonic method. In addition, the polysaccharide components of extracted surface coat examination by high-performance liquid chromatography (HPLC) indicated that the sheath is highly hydrophobic and is not hydrated to slime or RPS in the culture medium. The glucosamine (1.261±0.02 mg/g) contained in capsule makes the capsule more adhesion than the sheath. Additionally, the optimal extraction methods obtained from this study can be effectively applied to other cyanobacterial and green algal species whose surface coats are mainly capsule and sheath. This study may provide technical support for further revealing the biological function of cyanobacterial and algal surface coat.