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Phaeodactylum tricornutumhave garnered significant attention for its ability to accumulate fucoxanthin (Fx) and eicosapentaenoic acid. However, its limited biomass in autotrophic culture becomes a restrictive factor for the large-scale production of Fx. Herein, 6-benzylaminopurine and 5-aminolevulinic acid at 1.0 mg/L, were proved conducive for promoting the Fx and biomass accumulation. Under the combined effect of the two, Fx and peaked at 36.01 mg/L after 10 d of cultivation, at which point biomass was 1.50 g/L and Fx content was 24.01 mg/g. Meanwhile, EPA production was elevated to 22.73 mg/L. Such combined effect was also found in regulating the transcriptional levels of Fx biosynthesis-related genes by the two plant hormones. This study demonstrates that Fx and biomass can be enhanced easily and efficiently under the combined effect of these two plant hormones, enabling massive Fx production in the autotrophic system.