In order to study the role of sufficient phosphorus (P) in biodiesel production by microalgae, Phaeodactylum tricornutum were cultivated in six different media treatments with combination of nitrogen (N) sufficiency/deprivation and phosphorus sufficiency/limitation/deprivation. Profiles of N and P, biomass, and fatty acids (FAs) content and compositions were measured during a 7-day cultivation period. The results showed that the FA content in microalgae biomass was promoted by P deprivation. However, statistical analysis showed that FA productivity had no significant difference (p = 0.63, >0.05) under the treatments of N deprivation with P sufficiency (N-P) and N deprivation with P deprivation (N-P-), indicating P sufficiency in N deprivation medium has little effect on increasing biodiesel productivity from P. triornutum. It was also found that the P absorption in N-P medium was 1.41 times higher than that in N sufficiency and P sufficiency (NP) medium. N deprivation with P limitation (N-P-l) was the optimal treatment for producing biodiesel from P. triornutum because of both the highest FA productivity and good biodiesel quality.