Juglans mandshurica is a precious timber and economic tree species in Northeast China. Because of the reproductive characteristics of this species, such as long childhood and dioecious, the flowering period of male and female flowers is different, which directly affects the sexual reproduction process such as flowering and pollination. At present, the molecular mechanism of dioecious flowering of J. mandshurica is not clear. In this study, the cDNA sequence of a crucial gene involved in plant flowering, LEAFY (LFY) was cloned from J. mandshurica by homologous cloning, and the expression in different organs at various developmental stages, and the subcellular localization of the gene were analyzed. LFY in J. mandshurica was found to possess high homology (over 79%), and a similar structure and function to LFY in other species, and was cloned and named JmLFY (GenBank: KX364241). The results of fluorescence quantitative real-time PCR analysis revealed that JmLFY was expressed in different organs, and that expression increased significantly before the early development stage of staminate inflorescence, the anther formation and pollen dispersal stage, pistillate inflorescence sprouting and flower development stage, and the pollination and late stage of zygotic embryo development. Expression of the green fluorescent protein fusion JmLFY gene in onion epidermal cells was localized to the nucleus and cytoplasm. Our results indicate that the JmLFY gene is the key transcription factor involved in flower formation and development in J. mandshurica. These findings provide information that may help to overcome the bottleneck caused by the long juvenile period and asynchronous female and male flowers of the species in the future.