The study of biofilm formation has importance in a broad scale of industrial applications such as health and cosmetics products, food, water, paper mills, medical health and pharmaceuticals. Manual process for the optimization of different salts for growth of bacteria is labour intensive work and has low precision. In this study, we have used design expert software for biofilm yield optimization of a strain of Chroococcidiopsis cubana. C. cubana was exposed on outer surface of the monuments due to adverse environmental conditions and formed the blackish biofilm on it. This biofilm was grown in culture media and their DNA was extracted for strain confirmation. Strain was confirmed by 16s rRNA gene sequence using Sanger sequencing. The response surface method was used to optimize the concentration of two main components NaNO3 and K2HPO4 among various salts of BG 11 media. Response surface methodology (RSM) was studied by ANOVA coefficient estimation using F – test with very low probability value. The obtained goodness of fit was significant (R2 = 0.99). Estimation of coefficient was used for calculation of t and p – values and decided their significancy. The model having p value less than 0.05 was considered for optimization. Desirability of optimization was resolved from contour plot having concentration of NaNO3 = 13 and K2HPO4 = 6.50 for optimum biofilm yield. Using these parameters, the in vitro model of Chroococcidiopsis cubana resulted in a yield of 20 g/l biofilm in 10 days, hence can be successfully employed for in vitro cyanobacterial biofilm yield of Chroococcidiopsis cubana.