<p>The accelerating depletion of fossil fuels, rising fuel prices, and growing concerns about global warming linked to fossil fuel use have made microalgae an attractive feedstock for biodiesel production. In the current investigation, the potentials of a green microalgae <i>Scenedesmus dimorphus</i> (Turpin) Kützing for biodiesel production were explored through optimization of important nutrients and temperature under heterotrophic condition. First, effect of different concentrations of organic carbon and nitrogen sources on the cell growth, lipid productivity and fatty acids profile of a fast-growing microalgal culture, <i>S. dimorphus</i> (Turpin) Kützing was investigated. Upon optimization of glucose and nitrogen in BG11 medium, effect of different temperatures on the biomass and lipid productivities and fatty acids profile of <i>S. dimorphus</i> was investigated. Biomass and lipid productivity of heterotrophic culture under optimized conditions of carbon and nitrogen sources were 0.31 gL^− 1day^− 1 and 56.4 mgL^− 1day^− 1 respectively. These values were respectively 15.5-fold and 18.4-fold higher than the value obtained in photoautotrophic culture. Under optimized conditions of nutritional sources and temperature, the maximum biomass of 0.33 gL^− 1day^− 1 and lipid productivity of 58.74 mgL^− 1day^− 1 were obtained at temperature of 30&#xa0;°C. The fatty acids profile showed that both had mainly C₁₆ and C₁₈ FAs. These cultivation modes demonstrated that heterotrophic conditions enhanced biomass and lipid productivity of <i>Scenedesmus dimorphus</i> (Turpin) Kützing for biodiesel production. The lab-scale findings of current study provide strong basis for use of heterotrophic conditions to grow this strain for production of large amounts of biomass and lipid necessary for commercial application.</p>