ABSTRACT The daily rhythm affects a series of physiological functions in crustaceans. To study its effect on the physiological function in Eriocheir sinensis, a crustacean species of high economic value, we analyzed the hemolymph transcriptome during the daily rhythm by high-throughput sequencing. We sampled the hemolymph from crabs at four time points in a single day (06:00, 12:00, 18:00, and 24:00 h) and identified 3,01,661 and 1,03,998 transcripts and unigenes, respectively; some of the unigenes were annotated as core clock genes. Moreover, 15,564 differentially expressed genes (DEGs) were divided into nine different clusters. Functional enrichment analysis of DEGs indicated that the molting, metabolism, and immunity processes in E. sinensis were impacted by its daily rhythm. In addition, we mapped the DEGs involved in the daily entrainment pathway. To the best of our knowledge, this is the first comparative transcriptome analysis of crustacean hemolymph during the day–night cycle, and provides multi-level information for unraveling the finer regulatory effects of the daily cycle in crustaceans.