Abstract: Dendrobium nobile was transformed by ovary-injection of Agrobacterium. After artificial pollination in 10, 20, 30, 40, 45, 50 and 60 d, EHA105(pCAMBIA1301) harboring both β-glucuronidase gene (GUS) and hpt was injected into the ovary. The seeds of split fruit of D. nobile were cultured on the N16 culture medium by aseptic processing and detected by GUS expression. Forty-five days after cultured, the protocorms were diverted to N16 medium with hygromycin to gain hygromycin-resistance protocorms, then four randomly selected hygromycin-resistance protocorms were detected by PCR analysis. The study showed that ovary-injection of agrobacterium had harmful effects on the fruit development and seed formation. Ovary-injection of agrobacterium after artificial pollination 45 d had the highest efficiency, 18% seeds from the injected position showing GUS expression. Eleven hygromycin-resistant plants were gained in this study, and four randomly selected hygromycin-resistant plants were detected by PCR analysis and all of them were positive. The results showed that ovary-injection of agrobacterium transformation had advantages in genetic transformation of orchids.