Phaeodactylum tricornutum, a unicellular diatom, is considered a potential feedstock for the production of biofuel and a promising producer for high-value products eicosapentaenoic acid and fucoxanthin. However, a high-efficient cultivating strategy to achieve commercial production of triacylglycerol (TAG) from the diatom is an urgent demand. In this study, we optimized the content and ratio of nitrate and tryptone in the medium to enhance biomass and TAG accumulation simultaneously. Growth with tryptone as the sole nitrogen gave rise to the lowest cell density but the highest TAG content in P. tricornutum relative to nitrate, nitrite, ammonium or urea cultures. In 500 μM NaNO3 cultures, the growth of P. tricornutum increased with the increasing concentration (from 294 to 7056 μM nitrogen) of supplemented tryptone, however supplementation of high tryptone (≥882 μM nitrogen) decreased the neutral lipid content. Elevating nitrogen concentration from 294 to 882 μM via tryptone addition in 250 μM nitrate culture increased cell densities from day 6 to 10 and neutral lipid content on day 10. In particular, supplementing 588 μM nitrogen of tryptone in the 250 μM nitrate culture gave rise to the highest neutral lipid content on days 8 and 10 (increased by 109% and 62% relative to 500 μM nitrate-sole) with a comparable growth to that in 500 μM nitrate-sole culture from day 2 to 8. In conclusion, we optimized nitrate/tryptone ratio and found that a suitable tryptone addition to a relatively low nitrate culture was favourable to the biomass and TAG accumulation simultaneously in P. tricornutum.