In this study, the improved biomass (1.6 folds) and lipid (1.3 folds) productivities in Synechocystis sp. NN using agro-industrial wastes supplementation through hybrid response surface methodology-genetic algorithm (RSM-GA) for cost-effective methodologies for biodiesel production was achieved. Besides, efficient harvesting in Synechocystis sp. NN was achieved by electroflocculation (flocculation efficiency 97.8±1.2%) in 10min when compared to other methods. Furthermore, different pretreatment methods were employed for lipid extraction and maximum lipid content of 19.3±0.2% by Synechocystis sp. NN was attained by ultrasonication than microwave and liquid nitrogen assisted pretreatment methods. The highest FAME (fatty acid methyl ester) conversion of 36.5±8.3mg FAME/g biomass was obtained using titanium oxide as heterogeneous nano-catalyst coupled whole-cell transesterification based method. Conclusively, Synechocystis sp. NN may be used as a biodiesel feedstock and its fuel production can be enriched by hybrid RSM-GA and nano-catalyst technologies.