In this work, a water-stable, pyrene-functionalized Hf-based mixed-ligand metal-organic framework (MOF) with UiO-66 (UiO = University of Oslo) framework topology was designed and synthesized for the fluorimetric detection of uric acid (UA) in aqueous medium. The free pyrene-functionalized ligand changes its conformation (monomer to excimer) upon framework formation, which was confirmed via fluorescence spectral studies. The MOF material showed a fast, selective, sensitive (detection limit 1.4 μM), and ratiometric fluorescence response toward UA in pure aqueous medium. During sensing, a further conformational change (excimer to monomer) of the ligand molecule was observed within the MOF. These dual conformation changes helped us to utilize the MOF material for the sensing of UA. Detailed experimental studies showed that the sensing mechanism involves hydrogen-bonding interactions between UA and the framework ligand, which is responsible for the change in conformation and fluorescence emission properties of the pyrene-functionalized ligand molecule. The MOF probe could be reused up to five cycles of sensing events. The MOF material was also utilized for the detection of UA present in serum and urine samples. Moreover, MOF-coated paper test strips were developed for the on-site fluorimetric detection of UA.