Abstract: The objective of the study was to isolate phenolic compound from the Sargassum hystrix crude extract by sequential solvent extraction using ethyl acetate, dichloromethane and butanol. In vitro antioxidant activity of fractionated cytoplasmic and membrane bound extracts were investigated. In addition to total phenolic content, the antioxidant activity were studied using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, metal chelating ability and Singlet Oxygen Quenching (SOQ) activity. Singlet oxygen quenching activity was examined using linoleic acid as substrate, containing 100 ppm erythrosine as a photosensitizer. The results showed that the membrane bound fractions had higher total phenolic compound, radical scavenging activity, ferrous ion-chelating ability and singlet oxygen quenching activity than cytoplasmic fractions (p<0.05). Among the eight fractions from membrane bound and cytoplasmic extracts isolated by differential solvent extraction, aqueous and butanol fractions of membrane bound extracts showed the highest DPPH radical scavenging, metal chelating ability and singlet oxygen quenching activity. The IC50 and metal chelating activity of aqueous fractions were 0.27±0.02 mg mL-1 and 51.53±6.63%, respectively and butanol fractions were 0.33±0.03 mg mL-1 and 44.75±3.33%, respectively. The butanol and aqueous fractions could act as SOQ at 75 ppm and 80 ppm, respectively. These results suggested that butanol and aqueous fractions were the most potent radical scavenger, metal chelator and singlet oxygen quencher.