Early determination of abnormal levels of uric acid (UA), xanthine (XA), and hypoxanthine (HXA) in the body fluid and food stuff could be useful in preventing various diseases. In this paper, we have fabricated a self-standing hybrid film based on niobium (benzene 1,3,5-tricarboxylic acid) MOFs (Nb(BTC)MOFs) and carbon nanofibers (CNFs) and explored as an enzyme-free electrochemical sensor for simultaneous determination of these analytes in phosphate buffer (pH = 7). As-developed hybrid film has a high specific surface area that permits faster transport of analyte molecules across the electrode-electrolyte interfaces, whereas a large number of active oxygen functionalities in MOF matrix could promote the sensitivity of the hybrid sensor. The Nb(BTC)MOF@CNF modified glassy carbon electrode (GCE) sensor demonstrated excellent electrochemical responses to these purine derivatives. The diffusion-controlled electrooxidation reactions of these three analytes were noticed. The hybrid sensor exhibited a wide linear range of 5 to 2000 µM along with a low LOD of 70 nM, 76 nM, and 68 nM for UA, XA, and HXA, respectively. The present interference-free sensor showed high selectivity and good storage stability (40 days). Finally, the sensing performances of the Nb(BTC)MOF@CNF/GCE toward UA, XA, and HXA in real samples were found to comparable to those of high-performance liquid chromatography (HPLC) results.