Abstract: Background and Objective: Though polysaccharides are considered to constitute the main medicinal properties of herbal Spina Gleditsiae, the current research on their isolation and the anticancer/antioxidant activities remain as an ongoing challenge. The purpose of this study was to develop an isolation technique for polysaccharides of Spina Gleditsiae (PSG) and then determined their anticancer and antioxidant properties. Materials and Methods: This study developed an ultrasonic-assisted method for PSG isolation and then the anticancer activities of isolated PSG were determined using MTT cytotoxic test. Moreover, the antioxidant effects of PSG were evaluated in the cell antioxidant, total antioxidant activity, ABTS free radical scavenging and Iron ion reduction assays. Results: In this study, the method of PSG isolation was firstly developed and characterized with acceptable yield rate (1.85±0.12%) and time/resources consuming, which greatly facilitate the current and future study of PSG. Meanwhile, the growth inhibitory and antioxidant effects of PSG were firstly determine in esophageal/liver cancer and human embryonic kidney cells in vitro. The isolated PSG were able to inhibit the growth of EC109 and HepG2 cancer cells with IC50 at 32.19±2.07 and 34.17±3.3 μg mL–1, respectively. Moreover, PSG demonstrated the dose-dependent antioxidant, free radical scavenging and reductive activities. Conclusion: The PSG was confirmed as an active anticancer and antioxidant component of Spina Gleditsiae, providing a potential natural medicinal resource for further investigation and development. Moreover, the results of this study underpin the fundamental understanding of the medicinal activities of Spina Gleditsiae and support its current use in clinic of China.