Several types of green photosynthetic microalgae can grow through the process of heterotrophic growth in the dark with the help of a carbon source instead of the usual light energy. Heterotrophic growth overcomes important limitations in the production of valuable products from microalgae, such as the reliance on light, which complicates the process, raises costs, and lowers the yield of potentially useful products. The present study was conducted to explore the potential growth of green microalga Scenedesmus obliquus under mixotrophic and heterotrophic conditions utilizing Disperse orange 2RL Azo dye as a carbon source to produce a high lipid content and the maximum dye removal percentage. After 7 days of algal growth with dye under mixotrophic and heterotrophic conditions with varying pH levels (5, 7, 9, and 11), KNO3 concentrations (1, 1.5, 2, and 3 g/L), and dye concentrations (20, 40, and 60 ppm); dye removal percentage, algal dry weight, and lipid content were determined. The results showed that the highest decolorization of Disperse orange 2RL Azo dye (98.14%) was attained by S. obliquus in heterotrophic medium supplemented with glucose at the optimal pH 11 when the nitrogen concentration was 1 g/L and the dye concentration was 20 ppm. FT-IR spectroscopy of the dye revealed differences in peaks position and intensity before and after algal treatment. S. obliquus has a high concentration of oleic acid, which is enhanced when it is grown with Disperse orange 2RL Azo dye, making it ideal for production of high-quality biodiesel. In general, and in the vast majority of instances, heterotrophic cultivation is substantially less expensive, easier to set up, and requires less maintenance than mixotrophic cultivation. Heterotrophic cultivation allows for large-scale applications such as separate or mixed wastewater treatment along with biofuel production.