In the present investigation, formulate the new ecofriendly, low-cost commercial fertilizer for pilot-scale cultivation of a green microalgae, Tetradesmus obliquus RDS01, using bubble column photo bioreactor. Initially, we optimized the four different fertilizers, viz., urea—500 mg L−1, di-ammonium phosphate (DAP)—350 mg L−1, potassium—280 mg L−1, and sodium bicarbonate—165 mg L−1 using response surface methodology (RSM). The Tetradesmus obliquus RDS01 microalga was produced 350.25 mg g−1 of lipid, 240.35 mg g−1 of carbohydrate, 17.50 g L−1 of biomass, 98.52% of CO<inf>2</inf> utilization efficiency, 325 U mL−1 of acetyl CoA carboxylase enzyme, 5.73 mL g−1 of biodiesel, and 4.25 mL g−1 of bioethanol obtained in pilot-scale cultivation in optimized commercial fertilizer medium. The Acetyl CoA carboxylase enzyme protein molecular weight was observed at 64KDa on 15% SDS-PAGE. The 3D structure homology modeling of Acetyl CoA carboxylase enzyme protein structure was predicted and protein encodes with 498 amino acid residues. The Acetyl CoA carboxylase specific genes (aCCD) were 1230 bp isolated. The Tetradesmus obliquus RDS01 microalga–produced biodiesel was subjected to gas chromatograph mass spectrometer (GCMS) analysis and identified the 50 biodiesel compounds. The major biodiesel compounds, viz., Cis-8-methylicosanoate methyl ester (28.36%), Eicosanoic acid, methyl ester (12.36%), hexadecanoic acid methyl ester (13.15%), 14-heptadecatrienoic acid methyl ester (3.25%), tridecanoic acid methyl ester (10.96%), tetradecanoate methyl ester (11.35%), pentadecanoic acid methyl ester (12.57%). The presences of functional groups were identified from the biodiesel such as amides, alcohols, carboxylic acids, alkenes, alkenyl, carbonyl, and ketones groups using FTIR. The bioethanol was confirmed in the peak range 3.52 min by HPLC. Graphical abstract: (Figure presented.). © 2024 Elsevier B.V., All rights reserved.