Tilia amurensis is an economically valuable broadleaf tree species in Northeast China. The production of high-quality T. amurensis varieties at commercial scales has been greatly limited by the low germination rates. There is thus a pressing need to develop an organogenesis protocol for in vitro propagation of T. amurensis to alleviate a shortage of high-quality T. amurensis seedlings. Here, we established a rapid in vitro propagation system for T. amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture media in different stages. We found that Woody plant medium (WPM) was the optimal primary culture medium for mature zygotic embryos. The highest callus induction percentage (68.76%) and number of axillary buds induced (3.2) were obtained in WPM + 0.89 µmol/L 6-benzyladenine (6-BA) + 0.46 µmol/L kinetin (KT) + 0.25 µmol/L indole-3-butryic acid (IBA) + 1.44 µmol/L gibberellin A3 (GA₃). The multiple shoot bud development achieved the highest percentage (83.32%) in the Murashige and Skoog (MS) + 2.22 µmol/L 6-BA + 0.25 µmol/L IBA + 1.44 µmol/L GA₃. The rooting percentage (96.70%) was highest in 1/2 MS medium + 1.48 µmol/L IBA. The survival percentage of transplanting plantlets was 82.22% in soil:vermiculite:perlite (5:3:1). Our study is the first to establish an effective organogenesis protocol for T. amurensis using mature zygotic embryos.