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Colorectal cancer (CRC) is a leading causes of cancer-related mortality worldwide. Dysregulated expression of specific genes and non-coding RNAs contributes to CRC progression. This study investigated the expression and clinical relevance of CEACAM6, HOXA-AS3, and miR-29a in CRC, combining experimental data with bioinformatics analysis to assess their diagnostic and prognostic value. Tissue samples from 68 CRC patients (tumor and adjacent normal) were analyzed for CEACAM6, HOXA-AS3, and miR-29a expression by real-time PCR. Bioinformatics validation using TCGA, GEPIA, and ENCORI databases assessed expression, interactions, and assess clinical associations. CEACAM6 and miR-29a were significantly upregulated, while HOXA-AS3 was downregulated in CRC tissues compared to normal counterparts. Notably, miR-29a also showed elevated expression levels in patient serum samples (p < 0.05). Among the examined markers, CEACAM6 expression varied significantly with tumor differentiation status (p < 0.05). Serum levels of IL-6 were significantly increased in CRC patients (p < 0.05). Receiver operating characteristic (ROC) analysis demonstrated high diagnostic accuracy for miR-29a (AUC = 0.918). A significant positive correlation was observed between miR-29a expression in serum and tumor tissue (p = 0.038). Additionally, in silico analysis suggested regulatory interactions between HOXA-AS3 and the mRNAs of CEACAM6 and IL-6. CEACAM6, HOXA-AS3, and miR-29a may serve as promising biomarkers for early detection and prognosis of CRC. Integrating molecular profiling with bioinformatics validation provides a robust approach to uncover clinically relevant targets in CRC. The correlation between serum and tumor tissue miR-29a expression highlights its potential utility in liquid biopsy approaches for CRC.