<p>Microalgae are deemed as a potential solution for CO₂ capture and utilization. However, how to increase CO₂ capture and useful compounds production synergistically is still a challenge. In this study, a marine diatom <i>Phaeodactylum tricornutum</i> was induced under four conditions (ACHN: ambient CO₂ and 882 μmol L^-1 NO₃^-; ACLN: ambient CO₂ and 0 μmol L^-1 NO₃^-; HCHN: 5% CO₂ and 882 μmol L^-1 NO₃^-; HCLN: 5% CO₂ and 0 μmol L^-1 NO₃^-) for 24 h to explore the optimal conditions for CO₂ capture and eicosapentaenoic acid (EPA) production. HC increased F_v/F_m, POC (particulate organic carbon) and PON (particulate organic nitrogen) content, thereby promoting cell growth, while LN exerted adverse effects on these parameters, particularly with extended culture time. HCLN reduced F_v/F_m and Chl <i>a</i> content, but enhanced POC content, with growth unaffected. HC increased carbon capture rate by 151%, 100% and 111% at 6, 12 and 24 hours respectively. HC significantly enhanced EPA content and productivity, LN also induced more EPA, and HCLN led to highest EPA content and productivity. The optimal condition for EPA content and productivity was HCLN at 12 h, which reached 52.61 ± 2.62 mg g^-1 DW and 175.92 ± 7.96 fg cell^-1 h^-1, 101% and 177% higher than those under ACHN, respectively. This study provides physiological insights into CO₂ fixation and EPA accumulation in <i>P. tricornutum</i> under high CO₂ induction, demonstrates short-term physiological feasibility, and identifies promising induction conditions for CCUS-oriented cultivation strategies.</p>