Six subfractions (H2O eluted fraction, 20% ethanol eluted fraction, 40% ethanol eluted fraction, 60% ethanol eluted fraction, 80% ethanol eluted fraction and acetone fraction) of a 95% ethanol extract of Phyllostachys pubescens leaves were sequentially partitioned by column chromatography (macroporous absorption resin). For the first time, nine specific major flavonoids were isolated from this species. The antioxidant activity levels of these nine major flavonoids and six subfractions were evaluated using a radical-scavenging assay, including 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radicals and superoxide anion radicals. The total flavonoid content of the six subfractions was determined by colorimetry. Results indicated that the nine major flavonoids and three of the subfractions (20, 40,and 60% ethanol) exhibited powerful antioxidant activity. These three subfractions showed high levels of total flavonoid content. Our results suggest that the nine major flavonoids and three subfractions are promising natural alternatives to synthetic antioxidants as functional food ingredients and natural antioxidant.