Panax notoginseng (P. notoginseng) is a famous traditional Chinese Medicine and is widely used for functional food in folk. Panax notoginseng saponins (PNS) were the main active components in P. notoginseng. The composition and number of the side chain glycosyl in PNS show great influence on the activity of ginsenosides and can be changed greatly by enzyme conversion. With the development of biotechnology in food processing industry, the anti-tumor potential of PNS deserves attention. In this study, enzyme transformed PNS products (E-PNS) were prepared by recombinant thermostable β-Glucosidase Tpebgl3 from PNS. Through the optimization of enzyme catalytic condition, 380 mg E-PNS was obtained from the transformation of 500 mg PNS by 8000 U Tpebgl3 at 85 °C in 40 ml water for 2 h. The main components in E-PNS were notoginsenoside R2, ginsenoside Rh1 and Rg3. Both in vitro and in vivo results showed that the anti-tumor activity of E-PNS was better than PNS significantly. The IC50 value of E-PNS against 4T1 cells was 132 μg/ml. E-PNS could induce the cell apoptosis of 4T1 cells through death receptor and mitochondrial pathway in a dose and time-dependent manner. The phosphorylation of protein kinase B (AKT) in proliferation signaling pathway was inhibited by E-PNS. In tumor tissue, the pathological score decreased and angiogenesis were inhibited significantly by the treatment of E-PNS. The study suggested that Tpebgl3 has great potential and prospect in promoting the application of P. notoginseng as functional food or food addition in cancer prevention or treatment.