OBJECTIVE: To examine the role and diagnostic potential of miR-421 in prostate cancer (PCa). METHODS: Expression data and clinical information for miR-421 were obtained from the TCGA and Genotype-Tissue Expression (GTEx) databases. Experimental validation was performed at the cellular, blood, and tissue levels to confirm miR-421 expression and its association with clinicopathological features. ROC curves were drawn on the bioinformatic study using TCGA data. The target genes of miR-421 were predicted via four online databases, and protein interaction associations were analyzed for intersecting targets. Gene Ontology (GO) analysis was subsequently conducted to assess functional relevance. RESULTS: MiR-421 was significantly overexpressed in prostate cancer (PCa) patients, a finding validated in cell, blood, and tissue samples. ROC analysis on the bioinformatic study using TCGA data revealed that miR-421 reliably differentiated PCa tissues from normal tissues. Higher miR-421 expression was associated with an elevated Gleason score, advanced TNM stage, and metastasis. GO enrichment analysis indicated that the target genes of miR-421 were significantly related to diverse molecular functions. CONCLUSIONS: MiR-421 is a promising biomarker for diagnosing and predicting PCa.