To understand the effect of hypoxia on Chinese mitten crab Eriocheir sinensis, the transcriptomic profiles of hemocytes in the hypoxia (1.5±0.5) mg/L and the normoxia groups [(6.0±0.5) mg/L of DO] were obtained using Nova Seq 6000. Firstly, by splicing, annotating and screening the original data, in total, 128614 transcripts were obtained with an average length of 2634 bp. Meanwhile, using a 1.2-fold change (P<0.05) in expression as a physiologically significant benchmark. A total of 1687 differentially expressed genes were identified, including 899 up-regulated and 788 down-regulated genes. GO and KEGG analysis showed that many processes or pathways are involved in the crab against hypoxia stress, such as tricarboxylic acid cycle (TCA cycle), glycolysis, ECM-receptor interaction, gap junction, cell apoptosis, prophenoloxidase system (proPO), etc. Finally, ten selected genes from these processes or pathways were verified by qRT-PCR analysis. The results showed 6 up-regulated genes (integrin-β1, extracellular Cu/Zn-superoxide dismutase, phosphoinositide-3 kinase, phosphoglycerate mutase 2, PDGF/VEGF-related factor 1, and relish) and 4 down-regulated genes (innexin-7, 90 ku heat shock, mitochondrial ATP synthase subunit α, and aspartate aminotransferase) corresponded with the transcriptome analysis. This study preliminarily elucidated the molecular mechanism of hemolymph response to short-time hypoxic stress, and provided some valuable information for the further analysis of the mechanism under hypoxia stress in crabs.