Panax notoginseng is a commonly used Traditional Chinese Medicine (TCM) and has diverse pharmacological activities with triterpenoid saponins as its main active ingredient. In the cultivation of P. notoginseng, continuous cropping is a serious problem, which could induce reduced productivity, low tuber quality, and plant mortality. With unique advantages of easy control, relative stability, high yields, tissue culture is widely used in the protection of TCM resources. In this study, we screened one adventitious root line, multi-branched (MB) root induced from wild-type roots of P. notoginseng, with a high yield of total triterpenoid saponins (17.92mg/g). The morphology analysis showed that MB root had structure similar to that of wild-type roots, except for the highly branched phenotype. MB root also showed close gene expression levels and metabolite profiles, which were also similar to those of wild-type roots of Demonstration Park (S3Y). Pearson's correlation coefficient analysis confirmed the importance of key gene, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), and transcription factor, PnERF1, in regulating triterpenoid saponin biosynthesis in P. notoginseng. These results suggested that MB root possesses potential value in the large-scale cultivation of P. notoginseng.