Human intestinal trefoilfactor (hITF) cDNA has been transferred into protoplasts of Pleurotus ostreatus by electroporation. Integration of the hITF cDNA into the genome Pleurotus ostreatus was confirmed by PCR analysis. Then competitive enzyme linked immunosorbent assay (ELISA) was developed for detection and quantitative analysis of human intestinal trefoil factor. Results show that the highest hITF protein content in mycelia is 2 000～2 250 ng/g, about 1.5% of the total soluble protein. The transgenic Pleurotus ostreatus is proved to have good biological activity of preventing rats from the gastric ulcer induced by ethanol, in which both of polysaccharides and hITF have a direct effect on ulcer tissue.