The interspecific hybridization can take advantage of heterosis and combine the double parental traits most extensively, and is an effective strategy for improving species and stress tolerance breeding for cross-pollinated trees. Species of Fraxinus L. are important for landscaping, timber, and afforestation tree species. To solve problems of poor cold adaption associated with the introduction of Fraxinus sogdiana in Maoershan (Heilongjiang province, Northeast China), to improve pest resistance of Fraxinus mandshurica, and to take advantages of the characteristics of F. mandshurica and F. sogdiana, the interspecific hybridization of F. mandshurica × F. sogdiana was conducted. Tissue culture is the key technology for the promotion of excellent clones and is a powerful tool for germplasm conservation of endangered or valuable plant species. Basing on the embryo culture of hybridization seeds, a complete and efficient micropropagation technology of the Fraxinus mandshurica × Fraxinus sogdiana for its production was established by the uniform design, regression analysis, and verification tests for optimal conditions. In this micropropagation technology, induction rates of callus, adventitious shoot, and adventitious root were 83.5%, 65.9%, and 80.0%, respectively; the number of adventitious shoots was 12.5 produced per callus and the survival rate of the seedlings grew in the greenhouse could be 91.3% after acclimatization. These findings enriched the germplasm resources of Fraxinus species, supplied a micropropagation supporting technology for production of F1 hybrids with ideal traits and accelerated the genetic crossbreeding process of F. mandshurica.