[Objectives] This study aimed to study the tissue culture technique of Betula microphylla Bunge var. paludosa. [Methods] Taking seeds as explant, seedlings of B. microphylla were cultured. Then, stem sections with leaves were cut off and subjected to induction of clustered shoots. Finally, the rooting and transplanting of adventitious shoots were completed. Thus, the tissue culture system of B. microphylla was established. [Results] The natural seed germination induction medium was Murashige and Skoog (MS), and the germination rate was 31%. The most suitable shoot induction medium was WPM medium added with 1.0 mg/L of 6-benzylaminopurine (BA), and the multiplication coefficient was 13.7. The rooting medium was Woody Plant medium (WPM) added with 1.0 mg/L of indole-3-butytric acid (IBA), and the rooting rate was 100%. The transplanting substrate for tissue-cultured seedlings was composed of humus soil, vermiculite and perlite (V: V: V = 4: 3: 3), and the survival rate reached 88.75%. [Conclusions] The experimental materials are easy to obtain and preserve, and the proliferation is rapid. This study provides technical support for the rapid acquisition of the tissue-cultured seedlings of B. microphylla.