To figure out the key phenolic compounds accounting for the antioxidant effects of longan (Dimocarpus longan Lour.) seed extract, online fishing/knockout method, activity evaluation assays, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR–MS), and high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC–ESI–MS) analysis were used jointly for the first time. p-Coumaric acid–glycoside, (S)-flavogallonic acid, ellagic acid derivative, and methyl-ellagic acid glucopyranoside were first identified in longan seeds. In addition, our study revealed that ellagic acid as well as its derivative and p-coumaric acid–glycoside had important contribution to the potent antioxidant activity of longan seed extract, while gallic acid, corilagin, (S)-flavogallonic acid, methyl-ellagic acid glucopyranoside, and ethyl gallate showed very little contribution to the total antioxidant activity of longan seed extract. The combining use of the online fishing/knockout method, activity evaluation assays, FT-ICR–MS, and HPLC–ESI–MS analysis is a useful and simple strategy for screening of key bioactive compounds from complex extracts.