Background: Sophora tonkinensis Gapnep. is an important rare medicinal plant in China. There were only a few papers on the rapid propagation of S. tonkinensis through in vitro tissue culture, and still no report focuses on the quality analysis of in vitro tissue culture plantlets. Materials and Methods: The different concentrations of 6-benzylaminopurine (BAP), kinetin (KT), and indole-3-acetic acid (IAA) were used to establish and screen the optimal rapid propagation technology of S. tonkinensis by orthogonal test; the different concentrations of a-naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and ABT rooting power (ABT) were used to screen the optimal rooting technology. For quality evaluation of tissue culture plants, three different sites were chose to finish planting experiment. The leaf characteristics, radix ex rhizoma yield, and contents of matrine and oxymatrine were evaluated, respectively, to provide evidence of high yield and good qualities of tissue culture plants. Results: A large number of buds could be induced directly from epicotyl and hypocotyl explants on the Murashige and Skoog (MS) medium supplemented with 1.5 mg/l BAP, 0.5 mg/l IAA, and 0.5 mg/l KT; the best root induction medium was solid MS medium at half the macronutrient concentration supplemented with 1.0 mg/l NAA, 0.4 mg/l IBA, and 0.1 mg/l ABT. The rooting rate was 98%. All tissue culture plants showed normal leaf characteristics. Tissue culture plants from two sites possessed higher radix ex rhizoma yield and overall productivity of matrine and oxymatrine than those of seed plants. Conclusion: Tissue culture is a rapid, effective, and convenient propagation method for S. tonkinensis, and the quality of S. tonkinensis tissue culture plants meets the requirement of quality standard of China Pharmacopoeia (edition 2010), the crude drug from S. tonkinensis tissue culture plants will be suitable for substituting the crude drug from seed plants.