Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an attractive target for new cholesterol-lowering drug development. Here, we developed a method integrating ligand fishing, HPLC-Q-TOF-MS and interdisciplinary assay, aiming to explore potential PCSK9 inhibitors from mixtures rapidly and accurately. PCSK9 was expressed and purified firstly, and then the recombined PCSK9 was coated on the surface of magnetic beads (MBs). The PCSK9-immobilized MBs (PCSK9-MBs) were used for ligand fishing combined with HPLC and Q-TOF-MS/MS. Ginkgo biloba leaves (GBL), an herbal medicine widely used in Asia and Europe with good efficacy in treatment of hypercholesterolemia, were chosen as an illustration for ligand fishing. Two PCSK9 ligands were discovered from GBL and identified as kaempferol-3-O-rutinoside (1) and kaempferol 3-O-26″-(6‴-p-coumaroyl) glucosylrhamnoside (KCGR) (2). In order to verify fishing results and pick out more powerful PCSK9 inhibitors, molecular docking assay was further performed and KCGR was optimized to be an excellent PCSK9 inhibitor by the confirmation of affinity and activity bioassay. These results suggested that the developed approach could be applied to screen and analysis potential bioactive constituents from mixtures, which may improve the efficiency of drug discovery. Moreover, KCGR separated from GBL was expected to be a potential candidate of PCSK9 inhibitors.