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The successful monitoring of the anticancer drugs using nanostructured materials is very important but very challenging task. Beside this, uniform and ultra-small size of metal oxide nanoparticles is highly needed in order to enhance the catalytic activity which could result into the development of sensitive and selective electrochemical sensors for methotrexate (MTX). For this purpose, we have used a simple approach involving the polyaniline (PANI) as a sacrificing template for the growth of uniform and ultra-small Co3O4 nanoparticles by hydrothermal method. The structure, shape, composition and phase purity were studied by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD) and Fourier transform Infrared (FTIR) techniques. The average size of Co3O4 nanoparticles was below 50 nm. The cubic crystallography is confirmed for the Co3O4 nanoparticles. The electrochemical properties of PANI assisted Co3O4 nanoparticles for MTX drug was evaluated by cyclic voltammetry (CV) and linear sweep voltammetry (LSV) in Britton–Robinson buffer (BRB) of pH 3.5. The PANI assisted Co3O4 nanoparticles were found highly sensitive for the MTX drug and exhibited a linear range from 5-75µM of MTX and limit of detection for the modified electrode was estimated 1.98µM. The proposed electrochemical sensor is low cost, simple, highly sensitive and selective towards MTX detection. The synthetic methodology using the conducting polymer as a sacrificing template for the growth of controlled and ultra-small Co3O4 nanoparticles can be utilized for the wide range of electrochemical applications.