Novel, proton-conducting bio-membranes based on Ocimum sanctum leaf (known as holy basil) as a host material and various concentrations of ammonium formate (NH4HCO2) have been developed using the solution casting technique. The developed electrolytes are characterized by X-ray diffraction technique (XRD), differential scanning calorimetry (DSC), field emission scanning electron microscope (FESEM), thermo-gravimetric analysis (TGA), impedance spectroscopy, and linear sweep voltammetry (LSV) techniques. As per XRD analysis, a bio-membrane of 1 g of pure Ocimum sanctum with 0.9 M.wt% of ammonium formate shows a very high amorphous nature. The pore size of the highest proton-conducting bio-membranes (0.9 M.wt% of NH4HCO2) is found to be in the range of 1.079 to 4.392 μm, using FESEM. The thermal and chemical stability have been analyzed using thermo-gravimetric analysis (TGA). The biomaterial membrane 1 g of pure Ocimum sanctum (OS) with 0.9 M.wt% of ammonium formate has got highest proton conductivity of 7.21 ± 0.03 × 10−3 S.cm−1. The electrochemical stability of the highest proton-conducting membrane is found to be 2.36 V (LSV technique). A proton battery has been constructed using the highest proton-conducting membrane as an electrolyte. The constructed proton battery shows an open-circuit voltage of 1.84 V. The stability of the battery has been observed for 50 h. A proton-conducting membrane fuel cell (PEMFC) has been constructed using the highest-conducting proton membrane. The constructed fuel cell shows an open-circuit voltage of 652 mV. The performance of membrane fuel cell (PEMFC) has been studied using different loads.