•The first transcriptome for Tetraselmis subcordiformis' special hydrogen inducing behavior.•Overall changing pattern of transcription for microalgal H2 evaluation process.•Quantitative analysis unveiled the functions of different hydrogenase subunits.•Postulated mechanism for anaerobic dark induction of T. subcordiformis.•Global gene function analysis showed T. subcordiformis a good candidate for biofuel.It's the first comparative transcriptome study of Tetraselmis subcordiformis' H2 production with carbonyl cyanide m-chlorophenylhydrazone (CCCP) by de novo RNA-seq. The global analysis of the annotated genes and pathways unveiled it a good candidate for biofuels. Focusing on H2 production, the genes coding active center subunit (HydA) and assembly subunits (HydEF and HydG) of hydrogenase proportionally expressed during the induction but stopped after illuminated; on contrast, the gene of the active centre stabilization subunit (Hyd3) highly expressed during the H2 evolution, indicating its key role in the prolonged H2 production. The results also showed genes of HCP4, ppGpp phosphohydrolase and RpoD relating to the hydrogenase expression regulation. On the energy aspect, genes of CF-ATPases and ferredoxin-NADP reductase in chloroplast down-regulated while Mito-ATPase in mitochondria and pyruvate metabolism pathway up-regulated during the H2 production, consisting with our previous reports on physiological level.