A two-step strategy was employed to culture Dunaliella tertiolecta, an oleaginous unicellular green alga, combined by the salt stress and sodium azide intervention, to observe their effects on its lipid accumulation. When the algae cultured at different salt concentrations reached the logarithmic growth phase, sodium azide was added. The results showed that the addition of sodium azide significantly increased the lipid content and had no significant effect on cell biomass. The lipid yield and single cell lipid content under 50 μM sodium azide increased by 10.4% and 21.7%. Under the two-step culture condition, combining of the treatment of 50 μM sodium azide and 2.5 M salt stress, the total lipid productivity and single-cell lipid content were 10% and 70.5% higher than that of the control. It seemed that sodium azide and salinity might have a synergistic effect on the lipid accumulation of D. tertiolecta. It can be concluded that sodium azide is an effective inducer of lipid accumulation in D. tertiolecta, and two-stage cultivation is a feasible way to improve lipid accumulation in microalgae.