In order to understand the effect of light,carbon sources and concentration on the phaeodactylum tricornutum, the culture medium containing glucose,acetate and glycerol were chose, the biomass, cell concentration, biochemical substances and fatty acid content were tested. The results show that the phaeodactylum tricornutum is photoautotroph and mixotroph, which with selectivity for substrate concentration and organic carbon sources, the optimum concentration of glucose is 20 mmol/L, the optimal growth condition in 500 mL flask contains that the temperature is (25±1) ℃, the light intensity is 50 μmol/(m2·s), the pH is 7.5. Whatever the carbon sources are able to promote the biomass accumulation. When the cell concentration achieve to 2.5×106 cells/mL in the evening of the culture, the protein, sugar, chlorophyll and TOL content were analyzed. The biomass accumulation and biomass change in different growth conditions were determined. By ultrasonic extraction and Soxhelt extraction, the total of lipid reaches 20%(dry weight); The fatty acid content and composition with GC-MS were measured, and found the C16∶0 and C16∶1 are 34.8% and 11.9% (mass fraction) respectively, which are more than other components and could be used to extract bio-diesel.