We report on a new fluorimetric assay for β-galactosidase (β-gal) and faecal coliform bacteria that utilizes a long-wavelength dye, chlorophenol red-β-D-galactopyranoside (CPRG), that has been widely used for colorimetric assays. The novel feature of this new assay is the unexpected development of a large fluorescence response from liberated chorophenol red (CPR) upon complexation with poly-L-arginine (pR) in solution. The binding of CPR to pR occurs through the sulphonate group of CPR, causing formation of a charge-transfer complex and up to a 70-fold increase in emission intensity. A major advantage of the assay is the ability to utilize excitation and emission wavelengths in the red end of the spectrum, which avoids common interferences obtained when using UV-absorbing dyes such as 4-methylumbelliferyl-β-D-galactopyranoside. We provide data on the utility of CPRG as a fluorimetric reporter for both β-gal and Escherichia coli ATCC 25922 and demonstrate optimized reaction conditions for rapid and sensitive detection of E. coli at a level of 1 colony-forming unit (cfu)/10 mL after 12 h of culture followed by a 1-h assay, which is below the regulatory limit for testing of recreational water.