This study investigate the effect of various parameters on biomass and oil production of the isolated fungus Alternaria sp., also study the feasibility of using molasses as an alternative carbon source for oil production. Thus, eight independent variables (sucrose, yeast extract, KH2PO4, MgSO4.7H2O, KCl, FeSO4.7H2O, PH, Incubation days) were studied using the Plackett-Burman statistical design technique. Data revealed that sucrose, yeast extract and incubation days were found to be critical factors affecting lipid production. Therefore, they were optimized using a response surface methodology. The optimum conditions for this fungus were in g/l: (sucrose, 35, yeast extract, 2.33, KH2PO4 2, MgSO4.7H2O 0.25, KCL 0.25, FeSO4.7H2O 0.02) pH 6.5, 30 °C, static conditions and incubation period of 160 hours. Under optimized conditions, the fungus gave the maximum biomass of 11.8 g/l, lipid yield of 5.94 g/l and lipid content of 50.3 %, respectively. Fatty acid characterization of the produced oil by gas chromatography revealed that it contains 40.31, 26.11 and 33.58 % of saturated, monounsaturated and polyunsaturated fatty acids, respectively, with linoleic acid as the most dominant fatty acid. Molasses was utilized as a carbon source and it was supports both growth and lipid production with biomass of 19.3 g/l and lipid yield of 5.6 g/l. Its oil profile was containing 41.53, 33.14, 25.33 % of saturated, monounsaturated and poly unsaturated fatty acids. Consequently, this study provide new insights for the economical single cell oil (SCO) production with valuable profile using agro industrial waste as a substrate for growth for Alternaria sp
