Effects of different levels of factors including salinity (3, 6, 9), light intensity [20, 40, 60 μmol/(m²·s)] and temperature (10, 20, 30℃) on the growth, ARA and EPA contents in diatom Cyclotella sp. SHOU-B108 were studied by orthogonal experiments. The results showed that the highest biomass (dry weight) of Cyclotella sp. was obtained in the treatment of salinity 6, light intensity 40 μmol/(m²·s) and temperature 30℃ on 10th day. There were significant interactions among salinity, light intensity and temperature on the ARA and EPA contents in Cyclotella sp.. Temperature was the main factor influencing the ARA and EPA contents of the diatom. Lower salinity (3) combined with higher temperature (30℃) was beneficial to accumulating ARA but high light intensity (60 μmol/(m²·s)) was beneficial for accumulating EPA in the cells of Cyclotella sp.. It is therefore suggested that different fatty acids in the cell display different reflections of the same environmental change, and Cyclotella sp. SHOU-B108 cells with specific high nutritional value could be harvested by optimizing the salinity-light intensity-culture temperature combinations.