Phenylketonuria (PKU) is an autosomal recessive disorder, which is characterized by severe mental retardation, microcephaly and seizures. The symptoms of this disease can be prevented if detected soon after birth. Therefore, blood Phenylalanine (Phe) measurement is essential for the early diagnosis, treatment and dietary monitoring of PKU patients. The goal of this research was to introduce a rapid, precise and effective enzymatic colorimetric method using the recombinant Bacillus badius phenylalanine dehydrogenase (PheDH; EC 22.214.171.124) for the quantitative determination of Phe in dry-blood spots. This test was based on the enzymatic reaction of PheDH coupled with an artificial electron acceptor system composed of phenazine methosulfate (PMS) and iodonitro tetrazolium chloride (INT). This assay system contained PMS to transfer the electrons of NADH to INT, enabling the formation of formazan with an absorbance at 490 nm. Calibration curve was plotted and the experimental data were fitted by linear regression analyze. The regression equation and correlation coefficient (R2) were Y= 0.0109x + 0.032 and R2=0.996, respectively. This method showed a recovery in the range of 95.1%-102.6% and had the limit detection of 0.5 mg/dl for Phe. The between run coefficients of variation (CVs) mean was between 3.8% and 9.1%. The within-run CVs was between 8.5% and 18.6%. Furthermore, no interferences from other amino acids and Phe derivatives were observed. Altogether, we here presented a quick and reliable enzymatic colorimetric assay for application in newborn screening and monitoring of PKU patients.