AbstractWe have developed a simple, whole‐cell bioassay for the detection of bioavailable benzene, toluene, ethyl benzene, and xylenes (BTEX) and similar compounds. A genetically engineered E. coli strain expressing toluene dioxygenase (TDO) and toluene dihydrodiol dehydrogenase (TodD) was constructed, enabling the conversion of BTEX into their respective catechols, which were quickly converted into colored products by a horseradish peroxidase (HRP)‐coupled reaction. The intensity of the color formation was correlated to concentrations of the BTEX compounds. Under the optimized conditions, a detection limit (defined as three times the standard deviation of the response obtained from the blank) of 10, 10, 20, and 50 μM was observed for benzene, toluene, ethyl benzene, and xylene, respectively. The bioassay was selective toward BTEX‐related compounds with no interference observed with commonly used pesticides, herbicides, and organic solvent. The bioassay was very stable with little change in response over a 10‐week period. The excellent stability suggests that the reported bioassay may be suitable for field monitoring of BTEX to identify and track contaminated water and follow the bioremediation progress.