The electrocatalytic behavior of polyaniline (PAni) towards the electro-oxidation of L-ascorbic acid (L-AA) was studied by using cyclic voltammetry. PAni synthesized by oxidative polymerization method, and characterized by using high resolution-transmission electron microscopy (HR-TEM), scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDAX) confirms large globular structures, X-ray diffraction (XRD) analysis revealed that PAni with amorphous nature, whereas Fourier-transform infrared spectroscopy (FTIR) of PAni shows C-N stretching, X-ray photoelectron spectra (XPS) attributes the peaks C1s, N1s and O1s. UV–visible spectra revealed PAni is conducting and its Brunauer–Emmett–Teller (BET) surface area is 343.67 m2/g demonstrates it has high surface area and good adsorption ability. The PAni electrocatalyst showed an excellent selectivity towards L-AA in presence of different inorganic and biological expedients under similar conditions. A limit of detection (LOD) and limit of quantification (LOQ) were calculated found to be 5.1 µM and 17.2 µM respectively. Furthermore, the electrocatalyst activity towards L-AA were also validated for real sample analysis like lemon juice and vitamin-C tablet by quantifying the L-AA. The exceptionally higher conductivity and good electrocatalytic performance of PAni make it a promising material for sustainable energy generation and environmental protection without relying on metals or harmful chemicals.