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Detection of microbial contamination in water is imperative to ensure water quality. We have developed an electrochemical method for the detection of <i>E. coli</i> using bi-functional magnetic nanoparticle (MNP) conjugates. The bi-functional MNP conjugates were prepared by terminal-specific conjugation of anti-<i>E. coli</i> IgG antibody and the electroactive marker ferrocene. The bi-functional MNP conjugate possesses both <i>E. coli</i>-specific binding and electroactive properties, which were studied in detail. The conjugation efficiency of ferrocene and IgG antibodies with amine-functionalized MNPs was investigated. Square-wave voltammetry enabled the detection of <i>E. coli</i> concentrations ranging from 10¹–10⁷ cells/mL in a dose-dependent manner, as ferrocene-specific current signals were inversely dependent on <i>E. coli</i> concentrations, completely suppressed at concentrations higher than 10⁷ cells/mL. The developed electrochemical method is highly sensitive (10 cells/mL) and, coupled to magnetic separation, provides specific signals within 1h. Overall, the bi-functional conjugates serve as ideal candidates for electrochemical detection of waterborne bacteria. This approach can be applied for the detection of other bacteria and viruses.