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A chemiluminescence assay for detection of coliforms was developed based on reaction of -galactosidase enzyme from Escherichia coli with a phenylgalactosidase-substituted dioxetane substrate, leading to the generation of light at 530 nm. Polymixin B was added to increase cell membrane permeability. Light emitted from the reaction was measured in a luminometer and data correlated with counts of E. coli enumerated on McConkey Agar plates. Comparison of light intensity from cultures grown in nutrient broth and lactose broth (LB) showed LB to be a better choice as growth medium. Randomized block design comprising a two-way treatment structure with 6 dilution levels and 25, 50 or 55 l of E. coli culture indicated maximum sensitivity of the assay at 55 l of E. coli with 500 l of dioxetane substrate. An exponential increase in the intensity of light emitted was observed with exponential increase in numbers of E. coli. The detection limit for the assay was 102- 103 CFU in 30 min. Assay was able to detect presence of coliforms in all food and water samples tested as confirmed by plating samples on McConkey Agar. Enzyme-based chemiluminescence assay provides a simple and rapid method for detection of microbial contamination in food and water.