Rapid and in time diagnosis of phenylketonuria (PKU) in affected infants can help preventing the progress of mental and developmental disorders associated with the disease. Here we report the isolation of alkaliphilic Bacillus bacteria capable of producing high level of Phenylalanine dehydrogenase (PheDH) from soil. A new quantitative and rapid test for PKU diagnosis was then developed using the isolated bacteria. The bacterial cells were used to convert serum Phenylalanine to phenyl-pyrovic acid, which was measured quantitatively using spectrophotometer. Application of the method in diagnosis of PKU patients in parallel with the HPLC method produced essentially similar results. However, the cost per sample (10-20 cents per sample) using this new method was much less than that of the HPLC method. The method can be automated which is suitable for mass screening for PKU in populations in which funding is a limiting factor.