| Summary: | In this study, we constructed a recombinant <i>Escherichia coli</i> strain with different promoters inserted between the chromate-sensing regulator <i>chrB</i> and the reporter gene <i>luxAB</i> to sense low hexavalent chromium (Cr(VI)) concentrations (<0.05 mg/L); subsequently, its biosensor characteristics (sensitivity, selectivity, and specificity) for measuring Cr(VI) in various water bodies were evaluated. The luminescence intensity of each biosensor depended on pH, temperature, detection time, coexisting carbon source, coexisting ion, Cr(VI) oxyanion form, Cr(VI) concentration, cell type, and type of medium. Recombinant <i>lux</i>-expressing <i>E. coli</i> with the T7 promoter (T7-lux-<i>E. coli</i>, limit of detection (LOD) = 0.0005 mg/L) had the highest luminescence intensity or was the most sensitive for Cr(VI) detection, followed by <i>E. coli</i> with the T3 promoter (T3-lu<i>x-E. coli</i>, LOD = 0.001 mg/L) and that with the SP6 promoter (SP6-lux-<i>E. coli</i>, LOD = 0.005 mg/L). All biosensors could be used to determine whether the Cr(VI) standard was met in terms of water quality, even when using thawing frozen cells as biosensors after 90-day cryogenic storage. The SP6-lux-<i>E. coli</i> biosensor had the shortest detection time (0.5 h) and the highest adaptability to environmental interference. The T7-lux-<i>E. coli</i> biosensor—with the optimal LOD, a wide measurement range (0.0005–0.5 mg/L), and low deviation (−5.0–7.9%) in detecting Cr(VI) from industrial effluents, domestic effluents, and surface water—is an efficient Cr(VI) biosensor. This unprecedented study is to evaluate recombinant lux <i>E. coli</i> with dissimilar promoters for their possible practice in Cr(VI) measurement in water bodies, and the biosensor performance is clearly superior to that of past systems in terms of detection time, LOD, and detection deviation for real water samples.
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