Nicotinamide adenine dinucleotide (NADH) detecting biosensor of L-phenylalanine (Phe) was developed and demonstrated for diagnosis of phenylketonuria. This biosensor was comprised of L-phenylalanine dehydrogenase immobilized membrane, an ultra violet light-emitting diodes excitation system and a photomultiplier tube. The sensor was sufficient to clinical application of genotypic diagnosis for phenylketonuria.