Lysimachia davurica is an important medicinal and ornamental plant species. An efficient in vitro regeneration system on excised leaves of L. davurica was developed. The experimental conditions of tissue culture system were investigated based on the effect I of different concentrations and combinations of plant growth regulators (PGRs), explant orientations, leaf segments, and concentrations of activated charcoal. Two types of callus induction medium using Murashige and Skoog (MS) medium + 1.0 mg L-1 NAA + 0.5 mg L(-1)6-BA and 1.0 mg L-1 NAA + 1.0 mg L(-1)6 -BA had a callus inducing rate of 95.56%. The shortest time to callus appearance was 7 d. Proximal segments were better for callus induction than middle and distal ones. Placing leaf with abaxial side down on medium was better than adaxial side for inoculation. For the combinations of 6 -BA and NAA tested, MS + 0.5 mg L-16-BA and 0.1 mg L-1 NAA was best for callus proliferation, while MS + 1.0 mg L-16-BA and 0.1 mg L-1 NAA was optimal for callus differentiation and shooting. For the combinations of PGRs tested, the best rooting medium is 1/2 MS + 0.5 mg L-1 IBA + 0 mg L-1 NAA, with 99.89% rooting rate. On plantlets rooting, 1 g L-1 activated charcoal performed better than others (0, 2, 3, and 4 g L-1). The optimal transplanting medium was using vermiculite and humus as substrate in the proportion of 2:1 (v/v), with 97.78% survival rate. This protocol could be an efficient method for the micropropagation of L. davurica.