Research on the optimization of hydrolysis techniques on the removal of mucilaginous sheath branched filamentous Cyanobacteria has been carried out. The optimizations included the homogenizing process and addition of nitrogen in growth medium. The aim of the study is to analyze the process of hydrolysis based on the absence and presence of Nitrogen in culture medium and to analyze the effect of hydrolysis on removing the mucilaginous sheath. The hydrolysis treatment was performed on three strains of branched filamentous Cyanobacteria were used in this study: SO-130, SO-133 and SO-198 using five replications with homogenization process used five glass beads BioSpect 3.2 mm. The results were observed using microscopic observations with and without negative staining. The result showed that the mucilaginous sheath of three strains was disappeared after hydrolysis process that growth in medium with and without nitrogen. The DNA purity in all treatments was 0.81-1.19, indicating the absence of pure DNA. The DNA concentration were 1.63-6.47 ng/μL and have the same pattern in the three strains that was decreasing from control to treatment.