Single-cell oil has long been considered an alternative to conventional oil sources. The oil produced can also be used as a feedstock for biodiesel production. In this research, Morphological, microscopical, Biochemical tests and VITEK 2 YST ID card are used to identify most lipid screening yeast isolate named Y4. Many parameters (different Carbone sources, different nitrogen sources, PH, Incubation days, shaking, and statistic incubation) were studied using the optimization of one variable at time (OVAT).             On YED agar media, the isolated yeast Yarrowia lipolyticaY4 gave the maximum biomass of 4.15±0.04g/l, lipid yield of 0.86±0.03g/l and lipid content of 20.67±0.62%, respectively. Molasses was utilized as a carbon source and it was supporting both yeast growth and lipid production with two-fold increase biomass of 9.36±0.06g/l, lipid yield of 1.80±0.18g/l and lipid content of 19.31±1.81%. Also, the oil profile by gas chromatography was contain 36.17%, 56.54% and 7.26% of saturated, monounsaturated and polyunsaturated fatty acids after 7 days' incubation period at refrigerator and, contain 64.21%, 28.40%, and 7.33% of saturated, monounsaturated and polyunsaturated fatty acids after 30 days' incubation at refrigerator. Consequently, this work study new insights for the economical oil production with valuable profile using cheap agro-industrial waste molasses as a substrate for growth of marine Yarrowia lipolyticaY4yeast at low temperature 4°C for one week and one month.