Orthogonal experiments L₉(3⁴) were applied to investigate the effects of different salinity (6, 12 and 18), light intensity[10, 30 and 50 μmol/(m²·s)] and temperature (10, 20 and 30℃) combinations on the growth and cellular contents of arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in Chaetoceros sp. SHOU-B98. The results showed that salinity, light intensity, temperature and the interaction of the three factors had significant effects on growth and highly unsaturated fatty acid contents of the microalgae. Under salinity 12, light intensity 30 μmol/(m²·s) and temperature 30℃ combination and salinity 18, light intensity 50 μmol/(m²·s) and temperature 20℃ combination, the microalgae displayed the fastest growth and highest biomass since the 4th day (P<0.05). The best culture conditions for cell to accumulate different highly unsaturated fatty acids were also different. The optimal combined culture condition for Chaetoceros sp. SHOU-B98 cell to accumulate ARA was combination of salinity 6, light intensity 50 μmol/(m²·s) and temperature 30℃. While the optimal culture condition to accumulate EPA or DHA was combination of salinity 18, light intensity 50 μmol/(m²·s) and temperature 20℃. Therefore the suitable culture condition for microalgae Chaetoceros sp. SHOU-B98 with fast growth and high fatty acid nutrition value is the combination of salinity 18, light intensity 50 μmol/(m²·s) and temperature 20℃.