Abstract Galla Chinensis was a traditional Chinese medicine used as an ingredient in many prescriptions for the treatment of oxidant damage-associated symptoms in China. The current research on the isolation of polysaccharides from Galla Chinensis and their antioxidant activities still remain as an ongoing challenge. The purpose of this study is to develop an isolation technique for polysaccharides from Galla Chinensis and determine their antioxidant properties in vitro. An ultrasonic-assisted method of polysaccharides extraction from Galla Chinensis were firstly developed and the key parameters were optimized response surface analysis as crushing degree 100 mesh, ultrasonic time 20 min, and materials/liquid ratio 1: 40. The polysaccharides presented dose-dependent antioxidant and free radical scavenging effects in DPPH, ABTS, total antioxidant and Iron ion reduction assays. Moreover, it also demonstrated an effective protective effect for human under H2O2-induced oxidative damage on human embryonic kidneyfibroblast 293 and hepatoma 7721 cells by cell proliferation assay. The study firstly determined the antioxidant activities of polysaccharides from Galla Chinensis, thus providing a new natural resource for future investigation and development of the polysaccharides-based antioxidant drugs, health products or addictives.