D-Amino acid oxidase (DAAO) purified from goat kidney was immobilized covalently via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto poly indole 5-carboxylic acid (Pin5-COOH)/zinc sulfide nanoparticles (ZnSNPs) hybrid film electrodeposited on surface of an Au electrode. A highly sensitive D-amino acid biosensor was constructed using this enzyme electrode as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The biosensor showed optimum response within 3 s at pH 7.5 and 35 degrees C, when polarized at 0.15V vs. Ag/AgCl. There was a linear relationship between biosensor response (mA) and D-alanine concentration in the range 0.001-2.0 mM. The sensitivity of the biosensor was 58.85 mu A cm(-2) mM(-1) with a detection limit of 0.001 mM (S/N =3). The enzyme electrode was used 120 times over a period of 2 months when stored at 4 degrees C. The biosensor has an advantage over earlier enzyme sensors that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective layer of poly indole-5-carboxylic acid film. The biosensor was evaluated and employed for measurement of D-amino acid level in fruits and vegetables. (C) 2012 Published by Elsevier Ltd.