A novel biosensor for determination of d-amino acids (DAAs) in biological samples by using an electrode based on immobilization of a thermostable d-Proline dehydrogenase (d-Pro DH) within an agar gel membrane was developed. The electrode was simply prepared by spin-coating the agar solution with the d-Pro DH on a glassy carbon (GC) electrode. An electrocatalytic oxidation current of 2,6-dichloroindophenol (DCIP) was observed at -100mV vs. Ag/AgCl with the addition of 5 and 20mmolL(-1)d-proline. The current response and its relative standard deviation were 0.15muA and 7.6% (n=3), respectively, when it was measured in a pH 8.0 phosphate buffer solution containing 10mmolL(-1)d-proline and 0.5mmolL(-1) DCIP at 50 degrees C. The current response of d-proline increased with increase of the temperature of the sample solution up to 70 degrees C. The electrocatalytic response at the d-Pro DH/agar immobilized electrode subsequently maintained for 80 days. Finally, the d-Pro DH/agar immobilized electrode was applied to determination of DAAs in a human urine sample. The determined value of DAAs in the human urine and its R.S.D. were 1.39+/-0.12mmolL(-1) and 8.9% (n=3), respectively.