Background: In the realm of tumor-targeted therapeutics, Polo-like kinases (PLKs) are a significant group of protein kinases that were found recently as being related to tumors. However, the significance of PLKs in pan-cancer remains systematically studied. Methods and materials: We integrated multi-omics data to comprehensively investigate the expression patterns of the PLK family across various cancer types. Subsequently, study examined the associations between tumor mutation burden (TMB), microsatellite instability (MSI), immune subtype classification, immune infiltration, tumor microenvironment scores, immune checkpoint gene expression, and the PLKs expression profiles within various tumor types. Furthermore, using our mRNA sequencing data (TRUCE01) and four bladder cancer (BLCA) cohorts (GSE111636, GSE176307, and IMvigor210), We examined the correlation between the expression level of PLK and immunotherapy effectiveness. Next, Gene set enrichment analysis (GSEA) was evaluated to find that potentially enriched PLK signaling pathways. Utilizing TIMER 2.0, we conducted an immune infiltration analysis underlying transcriptome expression, copy number variations (CNV), or somatic mutations of PLKs in BLCA. Finally, mRNA expression validation of PLK1/3/4 by real-time PCR within 10 paired BLCA tissues, protein expression verification through the Human Protein Atlas (HPA), and PLK4 in vitro cytological studies have been employed in BLCA. Results: The expression of most of the PLK family members exhibits variation between cancerous tissues and adjacent normal tissues across various cancer species. Furthermore, the expression of PLKs demonstrates a significant association with immunotyping, infiltration of immune cell, tumor mutational burden (TMB), microsatellite instability (MSI), immunological checkpoint gene activity and therapeutic effectiveness in pan-tumor tissues. Additional investigation into the correlation between the PLK family and BLCA has revealed that the expression of the PLK genes holds substantial significance in the biological processes of BLCA. Furthermore, a notable association has been observed between the copy number variation, variant status, and the degree of certain immunological cell infiltration. Of note, the expression validation and in vitro phenotypic experiments have demonstrated that PLK4 has a significant function in promoting the BLCA cell proliferation, migration, and invasion. Conclusion: Collectively, based on various databases, our results highlight the involvement of PLK gene family in the formation of different types of tumors and identify PLK-related genes that may be used for therapy.