Leaf explants of Dracocephalum rupestre were utilized for regeneration employing direct and indirect differentiation pathways. Results revealed that the direct regeneration medium for leaf explants consisted of MS + 2 mg/l 6-BA + 0.1 mg/l KT + 0.05 mg/l NAA, yielding a differentiation rate of 63.46%. The induction medium for callus was composed of MS + 2 mg/l 6-BA + 0.1 mg/l 2,4-D + 0.5 mg/l IAA, resulting in an induction rate of 86.73%. For the differentiation of adventitious buds, the medium included MS + 2 mg/l 6-BA + 2 mg/l TDZ + 0.05 mg/l IAA, with a differentiation rate of 53.48%. The proliferation medium for adventitious buds generated through both pathways, comprised MS + 2 mg/l 6-BA + 0.05 mg/l NAA, with proliferation rates of 83.57% and 87.41%, respectively. The rooting medium suitable for both methods was 1/2MS + 0.1 mg/l NAA + 0.1 mg/l IBA, resulting in rooting rates of 83.69% and 79.15%, respectively. Comparatively, the direct differentiation pathway proved to be more efficient and time-saving, with leaf explants requiring 30 days less for regeneration compared with the indirect pathway. This study provides theoretical and technical support for subsequent genetic transformation research of Dracocephalum rupestre.