The marine diatom Phaeodactylum tricornutum is a polymorphological, ecologically significant, and well-studied model of unicellular microalga. This diatom can accumulate diverse important metabolites. Herein, we cultured P. tricornutum in an internally installed tie-piece flat-plate photobioreactor under 14.5 m mol L−1 (high nitrogen, HN) and 2.9 m mol L−1 (low nitrogen, LN) of KNO3 and assessed its time-resolved changes in biochemical compositions. The results showed that HN was inductive to accumulate high biomass (4.1 g L−1). However, the LN condition could accelerate lipid accumulation in P. tricornutum. The maximum total lipid (TL) content under LN was up to 42.5% of biomass on day 12. Finally, neutral lipids (NLs) were 63.8% and 75.7% of TLs under HN and LN, respectively. The content of EPA ranged from 2.3% to 1.5% of dry weight during the growth period under the two culture conditions. Peak volumetric lipid productivity of 128.4 mg L−1d−1 was achieved in the HN group (on day 9). The highest volumetric productivity values of EPA, chrysolaminarin, and fucoxanthin were obtained in the exponential phase (on day 6) under HN, which were 9.6, 93.6, and 4.7 mg L−1d−1, respectively. In conclusion, extractable amounts of lipids, EPA, fucoxanthin, and chrysolaminarin could be obtained from P. tricornutum by regulating the culture conditions.