Abstract: In order to investigate the haploid breeding technique for unfertilized ovules of tobacco, using tobacco male sterile hybrid KRK26 as the test material, five types of ovules at development stage were selected and cultured on CM1, CM2, CM3, CM4 and CM5 media which were optimized based on H, HW and N₆ media. The in vitro haploid induction technique of unfertilized ovules was studied. The results showed that the more mature the embryo sac was, the worse the effect of ovule proliferation induction would be. The ovules developed earlier, i.e. from the time when the flower's corolla was about to expose calyx to the time when the corolla was twice as long as the calyx, should be selected for culture. Among the five optimized media, medium CM1 had the best induction effect on the embryoid. Adjusting the agar concentration to 10 g/L on the basis of CM1 was beneficial to the inhibition of the embryoid vitrification. Transferring the embryoids to medium H without hormone could avoid the browning of the embryoids and was beneficial for the induction of regenerated buds. Transferring the regenerated buds to medium H or N₆ with IAA 10 mg/L for the culture was helpful for the rapid rooting of the regenerated buds and a large number of regenerated plants could be obtained. Flow cytometry results indicated that most of the regenerated plants were haploids. These results demonstrated that the culture system for inducing haploid plants from unfertilized ovules of male sterile tobacco was established.