An enzyme-based fiber optic D-amino acid biosensor is reported. It is based on coimmobilizing D-amino acid oxidase and horseradish peroxidase (HRP) to inert matrix bovine albumin via glutaraldehyde. A new fluorimetric substrate, thiamine, is used to indicate the sensing processes. The effect of pH, thiamine concentration, and temperature have been studied. Under optimized conditions, the detection limit is 0.3 mu g/ml, the linear range of D-amino acid concentration is from 0.8 to 15 mu g/ml with excellent reversibility and stability, and the recovery is between 97.8 and 110.0%. The sensor can be used for at least 3 weeks at room temperature. It is practicable to determine D-amino acid in serum directly with satisfactory results. (C) 1995 Academic Press, Inc.