High-level expression of D-amino acid oxidase (DAO) has been reported in Pichia pastoris by integrating the DAO gene under the control of the alcohol oxidase promoter (P-AOX1). However, the time taken to reach peak product concentration is usually long (similar to 43 h), and cultivation requires tight regulation of methanol feeding. In this paper, we describe the expression of His-tagged DAO (HDAO) in P. pastoris using the glyceraldehydes-3-phosphate dehydrogenase promoter (P-GAP). The maximal level of HDAO expression using the P-GAP integrant is attained in 13 h and is equal to that obtained using the P-AOX1 integrant in 43 h. We also explored the possibility of secreting HDAO in P. pastoris. In-frame fusion of Saccharomyces cerevisiae alpha-factor secretion signal under a P-GAP or P-AOX1 resulted in low-level secretion of active HDAO, which was not of practical use. The intracellularly expressed HDAO under P-GAP was purified by agar-based affinity support and then immobilized on Amberzyme oxirane resin. The immobilized HDAO, with specific activity of 75 U g(-1) (wet weight), could be recycled more than 14 times without significant loss of activity. The data suggest that intracellular production of HDAO under P-GAP, followed by affinity purification and immobilization on oxirane resin, may serve as an effective process for the manufacture of immobilized DAO for industrial application.