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The methanol extract of black bamboo (Phyllostachys nigra Munro) leaves showed significant radical scavenging activity in an ABTS+-based assay. The high-performance liquid chromatography (HPLC) coupled to an online ABTS+-based assay system was used to identify and quantify the antioxidant principles from bamboo leaves. Antioxidant compounds were isolated from the ethyl acetate soluble fraction through the activity profiles based on the online ABTS+ assay, and identified as isoorientin (1), orientin (2), vitexin (3), luteolin 6-C-(6-O-trans-caffeoylglucoside) (4), vittariflavone (5), and tricin (6) using NMR and HPLC-ESI/MS data. Antioxidant activity levels of the isolated isoorientin (1), orientin (2), and luteolin 6-C-(6-O-trans-caffeoylglucoside) (4) were 33.10, 30.96, and 17.11 mM for a 50% reduction of the ABTS+ radical, respectively. This HPLC online ABTS+-based assay system was a rapid and efficient method for measuring antioxidants’ activity-guided purification from natural resources.