As a kind of carotenoid which existed in various marine microalgae, fucoxanthin had many biological activities including scavenging free radicals, anti-cancer, anti-obesity and other physiological activities, especially in anti-tumor and slimming effect. Phaeodactylum triconutum (P. tricornutum) had the capability to accumulate plenty of fucoxanthin, while the environment and nutritional factors have a significant effect on the growth of microalgae and the content of fucoxanthin. In this study, effects of different salinity (20, 25, 30), pH values (7.5, 8.0, 8.5) and nitrogen source types (urea, ammonium bicarbonate) on the OD value, cell concentration, dry cell weight, fucoxanthin content of P. tricornutum were investigated in outdoor mass culture. The results showed that maximum cell concentration was 6.01×106 /mL, which was obtained in the culture medium with salinity of 20. There was no significant effect of different salinity on the increase of fucoxanthin content of P. tricornutum. Optimal pH value of the culture medium for P. tricornutum was 8.0. Maximum cell concentration and fucoxanthin content were 5.8×106 /mL and 17.79 mg/g, respectively. Compared with ammonium bicarbonate, urea as a nitrogen source was more suitable for the growth of P. tricornutum and the final cell concentration reached 4.1×106 /mL. However, in terms of fucoxanthin accumulation, ammonium bicarbonate as nitrogen source was more advantageous since the maximum fucoxanthin content reached 9.96 mg/g. Therefore, the optimum culture conditions in open raceway pond for P. tricornutum were summed up to be salinity of 20, pH 8.0 and ammonium bicarbonate as the nitrogen source.