In this approach, vanadium pentoxide nanorods (V 2 O 5 NRs) were prepared by utilizing a facile wet-chemical approach (WCA) in an alkaline phase at low temperatures then applied to detect acetylcholine. Various optical techniques, such as Ultra-Violet spectroscopy (UV-Visible), Fourier Transform Infra-red spectroscopy (FTIR), Powder X-ray Diffraction (XRD), Field Emission Scanning Emission Microscopy (FESEM), X-ray Electron Dispersive Spectroscopy (XEDS) and X-ray Photo-electron Spectroscopy (XPS) were applied to analyse the V 2 O 5 NRs for structural, optical, functional, morphological and elemental analyses. Adjustment of the light weight fabrication of NRs onto Glassy carbon electrode (GCE) exhibited sensitive and selective acetylcholine sensor probe. Based on the stable current-voltage relationship, analytical sensing parameters like sensitivity, Linear dynamic range (LDR), Limit of detection (LOD), Limit of quantification (LOQ), durability and interference measurement of the fabricated sensor (GCE/V 2 O 5 NRs/BPM) for acetylcholine detection were evaluated and reported in this paper. Here, Nafion was used as a binding polymer matrix (BPM) for the fabrication of flat GCE with V 2 O 5 NRs. Acetylcholine calibration curve was found to be linear throughout in a broad range of concentration of target acetylcholine analyte. Calibration curve was employed to calculate the sensing parameters such as sensitivity (101.27 nAμM À 1 cm À 2 ), LOD down to 11.58 pM, LOQ (38.60 pM) and LDR (100 pM ∼ 100 μM) of the developed GCE/V 2 O 5 NRs/BPM sensor probe. The use of the facile WCA method for the synthesis of low-dimensional V 2 O 5 NRs is a good strategy for developing an un-doped nanomaterial based sensor for enzyme-free bio-molecule recognition and detection via this electrochemical approach. The developed (GCE/V 2 O 5 NRs/BPM) sensor probe could be used to selectively detect acetylcholine in real biological samples by electrochemical method with satisfactory outcomes.