The invention discloses a quantitative detection kit for of phenylalanine. The kit comprises phenylalanine dehydrogenase, coenzyme Beta-NAD, coenzyme FMN, long-chain aliphatic aldehyde, FMN-NADH oxidoreductase, bacteriofluorescein, a Gly-NaOH buffer with a concentration of 0 .05 mol / L and a pH value of 10.0 and a K2HPO4-KH2PO4 buffer with a concentration of 0.1 mol / L and a pH value of 7.0. The invention also discloses a quantitative detection method for phenylalanine. The method comprises the following steps: subjecting phenylalanine and Beta-NAD to a dehydrogenation reaction under the action of phenylalanine dehydrogenase so as to produce Beta-NADH; allowing Beta-NADH and FMN to undergo electron transfer under the action of bacteriofluorescein and FMN-NADH oxidoreductase so as to generate blue-green light; and calculating the content of phenylalanine in a sample according to luminous intensity. The detection method has the advantages of high sensitivity, a wide linear range, high precision and accuracy, short detection time, capacity of high-throughput detection, etc.