A method for flow injection with chemiluminescence (CL) detection has been developed for the determination of proteins. It is based on the luminescence of the N-bromosuccinimide-fluorescein-protein system, where fluorescein is used as an energy transfer reagent in alkaline medium. The CL of the system is strongly enhanced by hexadecyl trimethyl ammonium bromide. Optimum conditions and possible mechanisms have been investigated. Under optimum experimental conditions, the linear range is from 0.4 to 40 µg·mL−1 for egg albumin, 0.2 to 20 µg·mL−1 for bovine serum albumin, and from 1 to 100 µg·mL−1 for bovine hemoglobin. The detection limits are 37, 62, and 240 ng·mL−1, respectively.