In this study, we report the synthesis and application of palladium-nickel nanoparticles decorated on functionalized-multiwall carbon nanotube Pd–Ni@f-MWCNT and employed as a sensitive non-enzymatic electrochemical glucose sensor. The composition and crystal structure was characterized by X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Raman spectroscopy. The surface morphology of Pd–Ni@f-MWCNT was investigated by a transmission electron microscopy (TEM). The electrochemical response of the Pd–Ni@f-MWCNT to glucose was examined via the cyclic voltammetry. Results revealed that the prepared electrode exhibits high electrocatalytic activity for the oxidation of glucose into gluconolactone. The amperometric response of the Pd–Ni@f-MWCNT electrode to glucose was investigated at a potential of 0.5 V that demonstrated an extended linear range of from 0.01 to 1.4 mM, very low detection limit of 0.026 μM, very high sensitivity at 71 μA mM−1 cm−2, as well as good reproducibility, high stability and applicability for the real sample analysis. The reliability of the developed sensor was verified by comparison with a commercially available glucometer. The obtained results indicate that Pd–Ni@f-MWCNT is a promising candidate for electrochemical glucose sensing. •The synthesis of novel conductive nanomaterials.•The fully characterization of novel bio-sensor.•High efficiency and stability of novel biosensor towards glucose.•Real time measurements of novel biosensor.