<p>Pathogenic microorganisms pose severe threats to global public health, demanding rapid and accurate detection strategies. This review comprehensively summarizes recent advances in zirconium-based metal–organic frameworks (Zr-MOFs) for pathogen separation and biosensing. Leveraging high stability, large surface area, and ease of binding to biomolecules, Zr-MOFs-functionalized magnetic composites enable efficient enrichment via specific aptamer binding or label-free adsorption. In biosensing, they serve as versatile platforms: enhancing electrochemical sensitivity with conductive materials/enzymes; offering excellent fluorescence performance via quenching properties, tunable ligands or doped metals; and enabling rapid visual detection through colorimetric characteristics and peroxidase-like activity. Ratiometric signals further enhance reliability in complex matrices, and dual-signal systems expand their application scope. In addition, multimode signal output platforms based on Zr-MOFs are evolving to meet the needs of different situations. These technologies are fast, sensitive, economical, and continue to evolve toward point-of-care testing, offering promising solutions to the challenges of pathogen diagnosis.</p> Graphical abstract <p></p>