Saccharomyces cerevisiae spores are dormant cells， which can tolerate various types of environmental stress. In our previous work， we successfully developed biological and chemical methods for enzyme immobilization based on the structures of S. cerevisiae spore wall. In this study， we employed biological and chemical approaches for the immobilization of D-xylose isomerase (XI) from Thermus thermophilus and D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens with yeast spores， respectively. The enzymatic properties of both immobilized XI and DPEase were characterized and the immobilized enzymes exhibit higher thermostability， broader pH tolerance， and good repeatability compared with free enzymes. Furthermore， we established a two-step approach for the bioconversion of D-glucose to D-psicose using immobilized enzymes. To improve the conversion yield， a multi-pot strategy was adopted for D-psicose production by repeating the two-step process continually. As a result， the yield of D-psicose was obviously improved and the highest yield reached about 12.0 %.