Proline dehydrogenase (ProDH; 1.5.99.8) plays an important role in specific determination of plasma proline level in biosensor and diagnostic kits. The goal of this research was to isolate and characterize ProDH enzyme from Iranian soil microorganisms.Screening of L-proline degradative enzymes from soil samples was carried out employing enrichment culture techniques. The isolate was characterized by biochemical reactions and specific PCR amplification. The target ProDH was purified and the effects of pH and temperature on the activity and stability were also tested.Among the 250 isolates recovered from 40 soil samples, only one strain characterized as Pseudomonas entomophila displayed the highest enzyme activity toward L-proline (350 U/l) than others. The enzyme of interest was identified as a ProDH and had K(m) value of 32 mM for L-proline. ProDH exhibited its best activity at temperature range of 25 to 35°C and its highest activity was achieved at 30°C. It was almost stable at temperatures between 25-30°C for 2 hours. The optimum pH activity of ProDH reaction was 8.5 and its activity was stable in pH range of 8.0-9.0 upto 24 hours. The enzyme was purified with a yield of 8.5% and a purification factor of 37.7. The molecular mass of the purified ProDH was about 40 kDa, and determined to be a monomeric protein.This is the first report concerning the ProDH production by a P. entomophila bacterium isolated from soil sample.