L-Phenylalanine (L-Phe) is one of the essential amino acids and is important for the human body because it is used in the biosynthesis of neurotransmitters. Phenylalanine dehydrogenase (PheDH) has been studied as a material for the L-Phe sensor. In particular, the accurate and simple determination of L-Phe concentrations in human samples is needed for the diagnosis and treatment of patients with phenylketonuria. Thermostable PheDH from the bacterium Thermoactinomyces intermedius is suitable as a sensor material; however, it is reactive to not only L-Phe but also L-tyrosine (L-Tyr). Thus, we attempted to improve its substrate specificity for the accurate measurement of L-Phe. In this study, we developed the PheDH mutant with improved substrate specificity by amino acid substitution. The PheDH mutant, in which the 290th asparagine residue was substituted with aspartic acid (N290D), is considered to form a new interaction in the substrate binding site and has a markedly reduced reactivity with L-Tyr compared with the wild-type PheDH; the relative activities (L-Tyr/L-Phe) of the wild-type PheDH and the PheDH N290D mutant were 64.0% and 1.5%, respectively. An enzymatic assay using this mutant was established to quantify the L-Phe concentration in human plasma. The relative errors were −10.3% to 7.4% when compared with the quantitative value determined by high-performance liquid chromatography/electrospray ionization mass spectrometry. Furthermore, the immobilized mutant showed a concentration-dependent reactivity with L-Phe and no reactivity with L-Tyr, which indicate its potential use as a sensor material.