The work aimed to enhance growth and biochemical substances in Scenedesmus obliquus by creating a growing suitable condition of enrichment culture with varied concentrations of KNO₃ as a nitrogen source. Kuhl medium was used for the cultivation of Scenedesmus obliquus with optimized nitrogen source concentration. Growth was determined by optical density (1.94 OD₆₈₀) on the 10^th day of culture, and protein (0.18 mg/ml) concentration was assessed with +50% conc. of KNO₃. The diminishment of KNO₃ (-50% conc. of KNO₃) showed the highest lipid output (31.08µg/ml) and the maximum carbohydrate content (0.2 mg /mL) on day 10 of the culture. Also, carotenoids increased by 20.51% over control. Total phenolic, flavonoid, and ascorbic acid contents were measured, recording 127.53 ±0.79 mg gallic acid equivalents/ g crude extract, 81.59 ±1.05 mg gallic acid equivalents/ g crude extract 149.27 ±1.04 mg.g⁻¹, respectively. In conclusion, this work highlighted those polyunsaturated fatty acids (PUFAs) of S. obliquus cultivated at (-50%conc. of KNO₃) that increased by 80%, compared to the control culture; linolelaidic and α-linolenic acids were the main dominant of PUFAs and constituted 17.84 % and 22%, respectively.