Systematically constructing a gene expression atlas of poultry tissues is critically important for advancing poultry research and production. In this study, the gene expression profiles of 9 major tissues of Lueyang black-bone chicken were successfully constructed by transcriptome sequencing technology. Through in-depth analysis of transcriptome data, a total of 10 housekeeping genes (HKGs) and 87 marker genes (MGs) were identified. Furthermore, by applying weighted gene co-expression network analysis (WGCNA), we delineated nine tissue-specific modules and 90 hub genes, offering novel insights into the regulatory networks underlying tissue-specific gene expression. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that HKGs were predominantly involved in maintaining fundamental cellular functions, with significant enrichment in pathways related to oxidative phosphorylation, cell cycle regulation, and DNA replication. MGs were closely associated with tissue-specific physiological functions, providing valuable insights into the molecular mechanisms governing tissue functionality. Notably, through multidimensional validation, EEF1A1 and FTH1 were confirmed to exhibit cross-tissue expression stability, establishing them as ideal reference genes for multi-tissue qPCR experiments in chickens. Additionally, we successfully identified tissue marker genes, including TNNT2, PIT54, SFTPC, and PGM1, which are specific to the heart, liver, lung, and breast muscle, respectively. The results of this study have important scientific value in expanding reference gene selection and elucidating tissue-specific molecular mechanisms, and provide solid theoretical support and technical guidance for poultry breeding improvement and production practice optimization.