Graphene oxide/carbon nanotube/poly (O-toluidine) (GO-CNT-POT) nanocomposite was prepared by a situ polymerization method and characterized by X-ray powder diffractometry (XRD) and scanning electron microscopy (SEM). The antibacterial activity of the obtained GO-CNT-POT nanocomposite was also evaluated against Gram positive bacteria Bacillus subtilis, Gram negative bacteria Escherichia coli and antibiotics (Amoxicillin) using the agar plate. The antibacterial study showed that the GO-CNT-POT was found to be most effective against both B. subtilis and E. coli respectively which was significant compared to the amoxicillin and the simultaneously GO-CNT-POT nanocomposite were fabricated onto glassy carbon electrode (GCE) using conducting coating binders by I-V technique, where the total analytical parameters were measured for the development of sensitive lead sensors (Pb(2+)). The GO-CNT-POT nanocomposite were deposited on flat-GCE (surface area: ∼0.0316cm(2)) to result in a sensor that has a fast response to selective Pb(2+) ions in buffer system. Features including sensitivity, detection limit, reproducibility, linear dynamic range, selectivity, and electrochemical performances were investigated in details with the GO-CNT-POT nanocomposite fabricated GCE electrodes. The calibration plot is linear (r(2): 0.9907) over the large concentration range (0.1nM to 1.0mM). The sensitivity and detection limit is calculated as 8.53164μAcm(-2)μM(-1) and 89.0 pM (at a signal-to-noise-ratio, SNR of 3) respectively.