We propose a bimetallic alloy composed of Pt and Ni embedded within laser-induced carbon nanofibers (Pt/Ni-LCNFs) as an enzyme-free transducer for the detection of glucose under physiological pH. Laser exposure on electrospun polyimide nanofibers, embedded with Pt and Ni precursors, facilitated not only the formation of LCNFs but also the generation of Pt/Ni nanoparticles with a radius of approximately 2 nm and a distinctive crystalline structure. X-ray photoelectron spectroscopy revealed the oxidation states of the laser-generated Pt/Ni and confirmed the formation of the Pt/Ni alloy nanocatalysts. Additionally, small-angle X-ray scattering has shown that the graphitic structures of the LCNFs strongly depend on the metal salt concentrations and molar ratio. Pt/Ni-LCNFs were exploited as enzyme-free electrodes for glucose sensing at physiological pH. The presence of Pt in the alloy enabled a low potential (−0.9 V for 20 s) in situ generation of highly localized OH− which facilitated glucose electrooxidation by Ni. Under optimized conditions, Pt/Ni-LCNFs achieved reliable glucose detection in physiological conditions (pH 7.4), with detection limit of 0.3 mM, linearity from 0.1 to 4 mM, and minimal interference from other electroactive species. Self-calibrated data acquisition strategy provided an excellent recovery rate (95 ± 10%) in diluted human serum. Furthermore, unlike enzyme-based sensors, the catalytic activity of Pt/Ni LCNFs was maintained after sterilization, highlighting their robustness and potential in biomedical applications and bioprocess monitoring.