There is an urgent need to develop a novel sensor that can quickly and accurately detect biological warfare agents (BWAs). A novel electrochemical sensor was specially designed and developed for Staphylococcus aureus as a potential BWAs without using any enzyme, antibody, or genetic material. Cyclic voltammetry (CV), linear resistance polarization (LRP), chronoamperometry, impedance spectroscopy, and Differential Pulse Voltammetry (DPV) techniques were used for fast and accurate detection of BWAs. Co-precipitation technique was used for the synthesis of CuO nanoparticles (NPs) for their use in the electrochemical sensor. The effect of temperature, stirring and addition of CuO NPs was thoroughly investigated. The storage, stability, and performance of electrochemical sensor was tested for 45 days. The current response of the electrochemical sensor was found linear with the correlation coefficient, R2 = 0.991. The major advantage of this novel electrochemical sensor is that immobilization of enzyme, antibody, or DNA is not required in sensing technology as the immobilization process makes biosensors a highly specific. The novel electrochemical sensor was very quick, accurate, non-specific, and provide reliable results in the qualitative and quantitative estimation of pathogenic bacteria as a potential BWAs. A novel electrochemical sensor was specially designed and developed for the detection of Staphylococcus aureus pathogenic bacteria as a potential BWAs without using any enzyme, antibody, or genetic material. Cyclic voltammetry, linear resistance polarization, chronoamperometry, impedance spectroscopy, and Differential Pulse Voltammetry (DPV) experiments were carried out for fast and accurate detection of BWAs. Storage, stability, and reusability of the electrochemical sensor was tested for continuous 45 days. The current response of the electrochemical sensor was found to be linear with the correlation coefficient, R2 = 0.991. The major advantage of this novel sensor is that immobilization of enzyme, antibody, or DNA is not required in sensing technology as the immobilization process makes biosensor a highly specific. The novel electrochemical sensor was proved to be very quick, accurate, non-specific, and provide reliable results in the qualitative and quantitative estimation of pathogenic bacteria as a potential BWAs.