Filamentous microalga Tribonema sp. has the advantages of highly resistance to zooplankton-predation, easy harvesting, and high cellular lipid content, in particular large amounts of palmitoleic acid (PA) and eicosapentaenoic acid (EPA). Therefore, Tribonema sp. is considered as a promising biomass feedstock to produce biodiesel and high-value products. In this work, we studied the effect of different concentrations of nitrogen (NaNO3: 255–3 060 mg/L), phosphorus (K2HPO4: 4–240 mg/L), iron ((NH4)3FeC12H10O14: 0.6–12 mg/L) and magnesium (MgSO4: 7.5–450 mg/L) on the biomass, lipid content, and fatty acid composition of Tribonema sp. FACHB-1786, aiming at enhancing cell lipid productivity. The growth of Tribonema sp. had a positive correlation with the concentration of magnesium, and the maximum biomass of Tribonema sp. (under the condition of 450 mg/L MgSO4) was 8.09 g/L, much greater than those reported in previous studies using the same and other Tribonema species under autotrophic conditions. Different nitrogen concentrations exerted no significant effect on algal growth (P > 0.05), but a higher nitrogen concentration resulted in a greater amount of lipid in the cells. The maximum volumetric productivities of total lipids (319. 6 mg/(L·d)), palmitoleic acid (135.7 mg/(L·d)), and eicosapentaenoic acid (24.2 mg/(L·d)) of Tribonema sp. were obtained when the concentrations of NaNO3, K2HPO4, (NH4)3FeC12H10O14, and MgSO4 were 765 mg/L, 80 mg/L, 6 mg/L, and 75 mg/L, respectively. This study will provide a reference for substrate optimization for Tribonema sp. growth and lipid production.