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The development of sensitive and selective analytical devices for monitoring glucose levels (GLU) in biological fluids is extremely important for clinical diagnostics. In this work, we produced a new composite based on NiFe2O4 and multi-walled carbon nanotubes (MWCNT), called NiFe2O4@MWCNT, to be applied as a non-enzymatic amperometric sensor for GLU. Both NiFe2O4 and NiFe2O4@MWCNT composites were properly characterized by XRD, SEM, FTIR, and Raman spectroscopy, which confirmed that the composite was successfully prepared. A glassy-carbon electrode (GCE) modified with NiFe2O4@MWCNT was investigated by cyclic voltammetry and applied for the amperometric GLU detection using batch-injection analysis (BIA). A linear working range between 50 and 600 µmol L−1 GLU with a significant increase in sensitivity (3-fold) in comparison with MWCNT/GCE was verified, with a detection limit of 36 µmol L−1. Inter-electrode measurements (n = 4, RSD = 10%) indicated that the sensor fabrication is reproducible. Furthermore, the proposed non-enzymatic sensor was selective even in the presence of other biomarkers found in urine. When applied to synthetic urine samples, recovery levels between 84 and 95% confirmed analytical accuracy and the absence of sample matrix effect. Importantly, the developed approach is simple (free of biological modifiers), fast (77 injections per hour), and practical (high-performance tool), which are suitable features for routine analyses. [ABSTRACT FROM AUTHOR]