Abstract: 1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first rate limiting enzyme in carotenoids MEP biosynthesis pathway of algae. In this study, the full-length cDNA sequence of dxs was gained by Next-generation sequencing technology and bioinformatics analysis was carried out to investigate the effects of six kinds of exogenous factors on the dxs gene expression of Phaeodactylum tricornutum. The result showed that the dxs cDNA was 2476 bp long, contained an open reading frame (ORF) of 2193 bp with highly conservative ThDP-binding and transketolase domain, encoding 730 amino acids. With 79.31 kD in relative molecular mass and 6.65 in the theoretical isoelectric point, The DXS protein that contains signal peptide, transmembrane domain, coiled helix and TM-helix, is a hydrophilic stable protein. Phylogenetic tree analysis indicated that the DXS protein was divided into two clades, plant and algae. Furthermore, DXS protein of plant were classified as DXS1and DXS2 while there were three clusters in algae, namely Bacillariophyta, Rhodophyta and Chlorophyta, with Phaeodactylum tricornutum belonging to Bacillariophyta. The results of induced expression showed that dxs gene of Phaeodactylum tricornutum was regulated by six exogenous factors, including MeJA, AA, ACS, PIF, DCMU and light quality. The expression of dxs gene was the highest under the treatment of 100 μmol/L MeJA, 62.5 mg/L AA, 1.6 mg/L ACS, 1.00 μg/L PIF, 0.2 mg/L DCMU and purple light, respectively. With the induction of MeJA and light quality, the expression changes of dxs and fucoxanthin content in Phaeodactylum tricornutum showed a high consistency, which indicated that dxs is one of the key genes that promotes the fucoxanthin enrichment in Phaeodactylum tricornutum. High quality genetic resources for the future use of metabolic engineering is provided in this study to improve the content of fucoxanthin in cells of algae, and also a theoretical basis for deeply exploring the molecular regulatory principle for the biosynthesis of fucoxanthin in Phaeodactylum tricornutum.