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<i>Rosa banksiae</i> Ait. (<i>R. banksiae</i>) is a traditional Chinese folk medicine and an ornamental plant. Most previous studies have focused on cultivation and utilization while there are few research papers on the pharmacological activity of <i>R. banksiae</i>. This study aimed to get a better understanding of <i>R. banksiae</i> by extracting polyphenols with fractionated extraction technology. The results showed that ethyl acetate phase (EAP) contained the most polyphenols, while water phase (WP) had the least. HPLC analysis indicated that rutin and luteolin-4′-<i>O</i>-glucoside existed in the EAP and butanol phase (BP), but quercetin was only detected in the EAP. Six phenolic compositions were not detected in WB. The antioxidant and anti-tumor abilities of the EAP and BP were excellent. The results revealed that <i>R. banksiae</i> possessed a great antioxidant capacity and was rich in polyphenols, thus indicating <i>R. banksiae</i> was suitable for being a natural antioxidant and an abundant source of polyphenols.