This study aimed to develop a simple, fast, specific, and eco-friendly and portable electrochemical nano biosensor for the detection of model targeted pathogenic bacteria from water. In this work, first we synthesized silver, gold, and iron oxide nanoparticles at pH, 5.2, 5.7, and 11.6 respectively. These all-metal colloidal homogenous nanoparticles are stabilized for more than two months at ambient temperature by using one-pot synthesis wet chemical and precipitation method. After synthesized these nanoparticles are characterized by different analytical techniques and finally these nanoparticles are deposited on glassy carbon electrode (GCE) electrode to developed two types of GCE modified electrochemical nano biosensors used for the detection of different strains of bacteria like Escherichia coli 25922 and Escherichia coli K12 from water by Voltammetry (Biopotentiostate) equipment at Cyclic voltammetry mode(CV).The cyclic voltammetry (CV) technique was used to measure the signals current of pathogenic bacteria as a function of applying potential. The developed biosensors were more sensitive, selective having lower limit of detection (LOD) of targeted pathogenic bacteria. In second step we characterized the nanoparticles by various characterization techniques to confirm the synthesized metal (gold, silver and iron) nanoparticles including, UV–visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), atomic force microscopy (AFM), energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FT-IR), x-ray diffraction (XRD) and zeta potential analysis (ZPA). UV-Vis, spectroscopy is the initial characterization technique to confirm the formation and stability of silver, gold & iron metal oxide nanoparticles and their surface plasmon resonance band (SPR) appeared at 392 nm, 528 nm, and 388 nm, respectively. TEM analysis confirmed the size and shape of the particles and the size of silver, gold, and iron oxide metal nanoparticles were 35, 25 and 18 nm in diameters respectively. The most of particles were spherical confirmed by TEM technique and particles having an excellent electrocatalytic application to detect microorganisms from water. EDS spectra provided information about the elemental analysis of silver, gold, and iron element as 43,79, and 62 % respectively presented in the synthesized silver, gold, and iron oxide nanoparticles. Zeta potential provided confirmed the surface charge of silver, gold, and iron oxide nanoparticles particles bearing a net negative (-ve) charge after being capped by L-cysteine molecules and sodium citrate used as a reducing/capping agent. Finally, this biosensor GCE modified with gold and iron nanoparticles is an innovative detection technique that is used to check the validity of the developed biosensor were apply for the rapid detection of targeted and non-targeted bacteria from real water samples. The signals current was linearly increasing while increasing the concentration of E. coli from 101 to 105 CFU/mL with the linear correlation of (r2 = 0.879) and (r2 = 0.875) of iron and gold nanoparticles-based modified biosensors. The proposed electrochemical biosensor detects the E. coli in a lower concentration of 101 CFU/mL and with the maximum range of 105 CFU/mL. Moreover, the electrochemical detection techniques offer rapid quantification at a very low cost and miniaturization of an instrument. The obtained results of this study demonstrate the first step for a portable biosensor for the routine monitoring of pathogenic bacteria from water and food. Hence, the biosensor was highly selective in detecting E. coli 25922 and E. coli K12 from water samples. In this work the developed potable biosensor modified nanoparticles are less time consuming and easily constructed methods used for onsite and also for lab scale.