[Objectives] Transcriptome sequencing was performed on the samples collected after blood discontinuous density gradient centrifugation to explore the key functions of the blood cells of Chinese Soft-shelled Turtle Pelodiscus sinensis and to find out the marker genes related to immune function. [Methods] Blood samples of P. sinensis were stained with DQ to further characterize and describe the cell types. We used Percoll separation method to isolate erythrocytes, lymphocytes and thrombocytes of P. sinensis from whole blood with densities greater than 60%, 20%﹣40% and 50﹣60% for bulk transcriptome sequencing, with 3 biological replicates per sample. [Results] DQ staining showed the morphological characteristics of 7 blood cells of P. sinensis (Fig. 1). Venn diagram analysis results showed that coexpressed and specifically expressed genes were screened based on expression levels between the two groups, among which 274, 628 and 295 specific genes were expressed in erythrocytes, lymphocytes and thrombocytes, respectively (Fig. 2a). 1 701, 1 570 and 1 280 differentially expressed genes (DEGs) were screened between lymphocyte and erythrocyte groups, between thrombocyte and erythrocyte groups, and between thrombocyte and lymphocyte groups, respectively (P–adjust < 0.001) (Fig. 2b). GO enrichment showed that the up-regulated gene of erythrocytes had lysozyme activity and transferase activity, and was involved in the regulation of active oxygen metabolism (Fig. 3a). The up-regulated differential genes of lymphocytes were related to cytokine activity, and participated in the immune response to regulate the signaling process of cell surface receptors and the response to viruses (Fig. 3b). The up-regulated differential genes of thrombocytes were mainly involved in myeloid leukocyte activation, granulocyte activation and defense response (Fig. 3c). KEGG enrichment showed that the differential genes of lymphocytes and erythrocytes were enriched to NF-κB signaling, EB virus infection, T cell receptor signaling pathway, etc (Fig. 4a). The differential genes of thrombocytes and erythrocytes were enriched to platelet activation and hematopoietic cell lineage (Fig. 4b). Differential genes of thrombocytes and lymphocytes were enriched to the IL-17 signaling pathway, the interaction of viral protein with cytokines and cytokine receptors in lymphocytes and thrombocytes cells (Fig. 4c). The cluster analysis results of stratified blood bulk transcriptome revealed important potential marker genes of three types of cells screened from single-cell transcriptome (Fig. 5). [Conclusion] We further explored the function of blood cells of P. sinensis turtle by using discontinuous density gradient centrifugation combined with bulk transcriptome sequencing. The high expression of specific genes in the three cell types provided a basis for cell marker gene screening and antibody development of P. sinensis.