Background <p>The phosphofructokinase-fructose bisphosphatases (PFKFBs) family is critical in tumors and the immune system. Nevertheless, the involvement of the PFKFB family genes in the immune microenvironment and prognosis of tumor patients has not been investigated using genomic and transcriptomic analyses.</p> Methods <p>We collected data from 33 cancers and comprehensively analyzed the PFKFB family genes for abnormal expression, genomic alterations, diagnostic and prognostic roles, subcellular localization, pathway enrichment, immune microenvironment, association with immune checkpoints, and drug sensitivity in the disease, as well as predicting patients’ immunotherapeutic response to ICI and targeting of small molecule drugs, and performed the above results by immunohistochemical staining, pathological sections, and experiments for validation. Finally, we experimentally confirmed the role of <i>PFKFB4</i> in bladder cancer.</p> Results <p>We found the differential expression and diagnostic biomarker value of PFKFBs family members in pan-cancer, and also found that they play a key role in pan-cancer in relation to the tumor microenvironment. PFKFBs are closely associated with common immune checkpoints, TMB and MSI, and PFKFBs family members can predict immunotherapy response in patients with a wide range of cancers. Finally, it was experimentally determined that knockdown of <i>PFKFB4</i> gene expression inhibited proliferation and invasion of bladder cancer cells.</p> Conclusions <p>Our study highlights the great potential of PFKFBs as prognostic and immunotherapeutic response biomarkers, which may pave the way for further investigation of the tumor infiltration mechanism and therapeutic potential of PFKFBs in cancer.</p>