Interest in the antioxidant activity of bamboo leaves is growing. To discover new sources of natural antioxidants, a TLC bioautography method combined with a new image processing method was developed to evaluate the antioxidant activity of leaf extracts from 15 different species of bamboo. The results showed that the methanolic extract of Bambusa. textilis McClure possessed the highest antioxidant activity among the selected bamboo species. To rapidly identify the antioxidant compounds, the crude extract of B. textilis McClure was analysed by HPLC-UV, and HPLC-micro-fractionation of the extract was carried out. Based on TLC bioautography-guided fractionation, three antioxidant fractions were isolated from B. textilis McClure by preparative chromatography. These three antioxidant compounds were identified as isoorientin 4''-O β-D-xylopyranoside, isoorientin 2''-O-α-L-rhamnoside and isoorientin according to their UV, MS, and NMR data. The proposed TLC screening method could therefore be an easy way to evaluate the antioxidant activity of bamboo leaves, and the results achieved should prove very helpful for promoting their utilization, as B. textilis McClure can be considered a promising plant source of natural antioxidants.