This research aims to evaluate the effect of molasses as a source of organic carbon on the growth and lipid profile of Dunaliella salina cultivated under different light regimes to determine its strategy and potential use to produce biodiesel. Two sources of nutrients (F/2 medium, F/2; molasses, M) and two light conditions (12 h light: 12 h darkness, 12L:12D) conformed four treatments (F/2 12L:12D, F/2 D, M 12L:12D, and MD) that were investigated with three replicates each. The concentration of microalgae produced by the groups under light conditions was more than double that of those grown in darkness. The highest biomass produced (46.91 ± 6.47 g L-1) was obtained in the F/2 medium with 12L:12D treatment, while the lowest (18.99 ± 0.78 g L-1) was in the M 12L:12D group. Regardless of the culture conditions, the lipids were mainly composed of the C16:0 and C18 fatty acids. The use of molasses as a culture medium produced the highest amount of C18:1, regardless of the light regime. Molasses promoted the highest production of fatty acids methyl esters. Depending on the culture medium, D. salina cells showed different percentages of saturated (SFA), monounsaturated (MFA), and polyunsaturated (PUFA) fatty acids. The highest amount of saturated fatty acids was obtained in F/2 12L:12D. The highest amount of polyunsaturated fatty acids was presented in the mixotrophic culture (molasses and light). The cultures grown with molasses were rich in monounsaturated fatty acids. An inexpensive industrial by-product, Cane molasses could be a low-cost feedstock for biodiesel in cultivating D. salina.