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Rapid and sensitive detection of pathogens is a key requirement for both environmental and clinical settings. We report here a colorimetric enzyme–nanoparticle conjugate system for detection of microbial contamination. In this approach, cationic gold nanoparticles (NPs) featuring quaternary amine headgroups are electrostatically bound to an enzyme [β-galactosidase (β-Gal)], inhibiting enzyme activity. Analyte bacteria bind to the NP, which releases the β-Gal and restores its activity, providing an enzyme-amplified colorimetric readout of the binding event. Using this strategy, we have been able to quantify bacteria at concentrations of 1 × 10² bacteria/mL in solution and 1 × 10⁴ bacteria/mL in a field-friendly test strip format.