Dracocephalum rupestre is an important medicinal plant. However, its leaf explant regeneration system has yet to be established. Here we investigated the impact of plant growth regulators on callus induction, differentiation, adventitious bud proliferation, and rooting using field planted leaves and tissue-cultured seedling leaves of D. rupestre as explants. We found that the optimal medium for inducing callus from field planted leaves was MS+1.0 mg·L⁻¹ 6-BA+ 0.1 mg·L⁻¹ 2,4-D+1.0 mg·L⁻¹ IAA, achieving an induction rate of 84.51%. For adventitious bud differentiation, the preferred medium comprised MS+3.0 mg·L⁻¹ 6-BA+0.5 mg·L⁻¹ TDZ+0.5 mg·L⁻¹ IAA, resulting in a differentiation rate of 66.37%. Similarly, for inducing callus from tissue-cultured seedling leaves, the optimal medium included MS+2.0 mg·L⁻¹ 6-BA+0.1 mg·L⁻¹ 2,4-D+0.5 mg·L⁻¹ IAA, with an induction rate of 86.73%. The medium conductive to adventitious bud differentiation consisted of MS+2.0 mg·L⁻¹ 6-BA+2.0 mg·L⁻¹ TDZ+0.05 mg·L⁻¹ IAA, yielding a differentiation rate of 53.48%. Furthermore, the appropriate medium for adventitious bud proliferation was MS+2.0 mg·L⁻¹ 6-BA+0.05 mg·L⁻¹ NAA, achieving a proliferation rate of 83.57%. The rooting medium was 1/2MS+0.1 mg·L⁻¹ NAA+0.1 mg·L⁻¹ IBA, resulting in a rooting rate of 86.97%. Tissue-cultured seedlings exhibited optimal growth in a mixed substrate of peat and vermiculite at a ratio of 1:1 (v/v). This study successfully established a leaf explant regeneration system for D. rupestre, providing valuable technical support for the conservation and rapid propagation of its germplasm resources.