Roselle (Hibiscus sabdariffa L.) belongs to the malvaceae family. Its calyx is used for making jellies, jams, and beverages. Despite it usefulness, there are few or no studies on the purification and structural identification of HAs and Roselle extracts. This paper therefore seeks to investigate the purification and structural identification of Hibiscus sabdariffa L. extracts, and to evaluate their antioxidant activities on DPPH and ABTS and anti-tumor activities on human cervical cancer HeLa cells of purified fractions. Two fractions (fraction 1 and fraction 2) from Roselle (Hibiscus sabdariffa L.) extracts were purified with C-18 Sep-Pak cartridge, followed by Sephadex LH-20 column and characterized by HPLC-ESI-MS/MS. The contents of the anthocyanins in fraction 1 and fraction 2 were 863.41 mg/g and 617.35 mg/g, respectively. Nine compounds were identified with HPLC-ESI-MS/MS, seven of which have not been reported before in Roselle. Two fractions and HAs-rich extracts purified with C-18 Sep-Pak cartridge showed high capacity on radicals scavenging and HeLa cells inhibition in a dose-dependent manner. The orders of both activities were: fraction 1 > fraction 2 > HAs-rich extracts. IC50 values of fraction 1 on DPPH and ABTS radicals were 17.14 +/- 2.24 g/mL and 85.91 +/- 6.72 g/mL, respectively. The cell inhibition ratio of fraction 1 against HeLa cells reached 83.67 +/- 3.07% at the concentration of 200 g/mL. Two fractions and HAs-rich extracts could be a good supplements for natural antioxidant and anti-tumor activities.