The World Health Organization states that every year more than 3.4 million human deaths occur as a result of waterborne diseases. Here, we reported an innovative and amino functionalized electrochemical biosensor for the rapid detection of Escherichia coli (E. coli). Functionalized Fe3O4 NPs developed the sensor with L-cysteine, and cyclic voltammetry (CV) was employed to detect the bacteria. The results from this work are the first step toward the development of a portable water sensor that addresses the challenges of conventional time consuming and more expensive laboratory-based analyses. The detection of the targeted bacteria was performed with different bacteria to check the validity of the developed biosensor. Hence, the biosensor was highly selective to detect the E. coli from tap water samples. The signals current were increasing linearly while increasing the concentration of E. coli with the ranging from 10(1) to 10(5) CFU/mL with the linear correlation of (r(2) = 0.879). The proposed electrochemical biosensor detects the E. coli in a lower concentration of 10(1) CFU/mL and with the maximum range of 10(5) CFU/mL. The results from this work demonstrate the first step in a portable biosensor for the routine monitoring of microbial contamination from water. (c) 2019 The Electrochemical Society.