<p>The spleen and head kidney are important immune organs in fish, and the trunk kidney, while primarily a urinary organ, also plays a significant role in immunity. However, the immunological similarities and differences among these organs have not yet been systematically elucidated in fish. This study investigates the composition of immune cells and gene expression characteristics in the spleen, head kidney, and trunk kidney of <i>Hemibarbus labeo</i> to reveal similarities and differences in their immune functions at both cellular and molecular levels. All three organs contain abundant red blood cells, lymphocytes, and monocytes, with granulocytes particularly prevalent in the head kidney and trunk kidney. A total of 255,871 transcripts and 112,051 unigenes were identified, including 13,739 differentially expressed genes. Genes expressed in the spleen, head kidney, and trunk kidney of <i>H. labeo</i> are enriched in pathways related to pathogen phagocytosis, antigen presentation, and cytokine secretion. However, the main immune-related pathways enriched in each organ differ. Compared to the head kidney and trunk kidney, the spleen shows higher expression levels of genes involved in cytokine-cytokine receptor interactions. In contrast, the head kidney exhibits higher expression of genes related to the hematopoietic cell lineage, the B cell receptor signaling pathway, and Fc gamma R-mediated phagocytosis. The trunk kidney expresses higher levels of genes involved in the leukocyte transendothelial migration pathway. Additionally, neutrophil marker genes such as <i>crp</i>,<i> cxcr2</i>,<i> and ncf1</i> are more highly expressed in the head kidney and trunk kidney than in the spleen, suggesting that neutrophil-mediated immune functions are more pronounced in these organs. This study provides a molecular foundation for future immunological research on <i>H. labeo</i> and offers valuable insights into the functional similarities and differences among these key immune organs.</p>