A new method in which luminol-hydrogen peroxide luminescent system is used has been proposed for the determination of the presence of protein. Since the catalytic activity of copper(II) for the chemiluminescent reaction between luminol and hydrogen peroxide decreased when copper(II) interacted with polypeptide linkage, this phenomenon was applied to the determination of protein. Determination of protein was carried out by a flow-injection method. The effects of reagent concentration, flow-rate, and reaction time on the analytical value were examined and the conditions for the determination of protein were established. Similar calibration curves were obtained for human serum albumin, bovine serum albumin, bovine serum α-globulin, and bovine serum γ-globulin. According to the present flow-injection method using chemiluminescent reaction, a small amount of protein could be conveniently and economically determined over a wide range of concentration, 7×10^−4–7×10^−2 g dm^−3, with the detection limit of 0.2 μg and at the rate of about 30 samples per hour. The present method was applicable to the determination of protein in serum.