The huge demand for the clinical diagnosis of diabetes mellitus has prompted the development of great-performance sensing platforms for glucose detection. Non-enzymatic glucose sensors are getting closer to their use in realistic applications. In this work, polyvinylpyrrolidone (PVP)-conjugated bimetallic Pt–Pd nanosuperlattices were synthesized precisely through a simple synthesis procedure, leading to controllable spherical morphologies with significantly fine and precise nanostructures in a size range of ∼3–5 nm by the reduction of Pt and Pd precursors in ethylene glycol, using an ultrasonic method. High-resolution transmission electron microscopy (HRTEM) measurements evidenced the formation of Pt–Pd bimetallic nanosuperlattices (BMNSLs). The superlattice-fringe patterns (111) of bimetallic Pt–Pd NSLs were identified in the HRTEM images, clearly showing their crystalline nature. The prepared material was used in the electrochemical oxidation of glucose using voltammetry analyses. The experimental evidence indicates that the Pt–Pd BMNSL modified glassy carbon electrode is effective for the selective amperometric detection of glucose in the presence of galactose, sucrose, fructose, lactose, and ascorbic acid. Moreover, its application in the detection of glucose in real serum and urine samples was assessed and good recoveries are achieved. The results show that a Pt–Pd bimetallic nanosuperlattice with high surface area, catalytic activity, and superior selectivity could be a promising material in the generation of novel electrodes for low-cost non-enzymatic glucose sensors.