Abstract: The hypothalamic-pituitary-ovarian (HPO) axis orchestrates a coordinated neuroendocrine system. Despite its significance, the intrinsic heterogeneity within the three constituents of the HPO axis in vertebrates and the intricate interactions among various cell types remain largely elusive. This study presents the inaugural unbiased, cell-type-specific molecular characterization of all three components of the HPO axis in adult Lohmann layer and Liangshan Yanying chicken. Within the hypothalamus, pituitary, and ovary, we identified 7, 12, and 13 distinct cell types, respectively. Our findings indicate that the PACAP, FSH, and PRL pathways may exert influence over the synthesis and secretion of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in the hypothalamus and pituitary. In the ovary, interactions between granulosa cells and oocytes involve pathways such as KIT, CD99, LIFR, FN1, and ANGOTL, orchestrating the regulation of follicular maturation. The SEMA4 pathway emerges as a potentially pivotal player in the HPO axis, serving as a shared pathway across all three tissues. Our analysis of gene expression profiles in the three tissues reveals that relaxin 3 (RLN3), gastrin-releasing peptide (GRP), and cocaine and amphetamine regulated transcripts (CART) may act as novel endocrine hormones, participating in the regulatory activities of the HPO axis through autocrine, paracrine, and endocrine pathways. Furthermore, differential analyses between Lohmann layer and Liangshan Yanying chicken indicate heightened expression of GRP, RLN3, CARTPT, LHCGR, FSHR, and GRPR in the ovaries of Lohmann layer, correlating with superior reproductive performance. In conclusion, this study furnishes a comprehensive understanding of the molecular dynamics with in the HPO axis, potentially advancing reproductive biology and therapies for ovary-related diseases.