Research on the hydrolysis technique to remove mucilaginous sheath of filamentous Cyanobacteria has been conducted. The research aims to know the effect of addition of nitrogen in medium and homogenization with glass beads on mucilaginous sheath removal of filamentous Cyanobacteria. Three strains of Cyanobacteria used in present study were Nostoc sp. SO-24, Cylindrospermum sp. SO-115, and Calothrix sp. SO-163. Each strain was grown in Blue-Green 11 (BG-11) medium with and without nitrogen content, then homogenized with 3.2 mm glass beads, and hydrolyzed. The result biomass was extracted to get the DNA. The result of hydrolysis process was examined through microscopic observation with and without negative staining. The result showed that addition of nitrogen and homogenization+hydrolysis process affected the mucilaginous sheath removal in all strains. Through this treatment, DNA concentration of SO-24 and SO-163 were higher than SO-115. Purity of DNA of all strains was low, ranging from 0.79-1.71. © 2019 Author(s).