14-3-3ζ protein, the key target in the regulation and control of integrin β3 outside-in signaling, is an attractive new strategy to inhibit thrombosis without affecting hemostasis. In this study, 4'--methylbavachalconeB (4--MB) in Psoraleae Fructus was identified as a 14-3-3ζ ligand with antithrombosis activity by target fishing combined with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. The competitive inhibition analysis showed that 4--MB targeted 14-3-3ζ and blocked the 14-3-3ζ/integrin β3 interaction with inhibition constant () values of 9.98 ± 0.22 μM. Molecular docking and amino acid mutation experiments confirmed that 4--MB specifically bound to 14-3-3ζ through LSY9 and SER28 to regulate the 14-3-3ζ/integrin β3 interaction. Besides, 4--MB affected the integrin β3 early outside-in signal by inhibiting AKT and c-Src phosphorylation. Meanwhile, 4--MB could inhibit ADP-, collagen-, or thrombin-induced platelet aggregation function but had no effect on platelet adhesion to collagen-coated surfaces in vivo. Administration of 4--MB could significantly inhibit thrombosis formation without disturbing hemostasis in mice. These findings provide new prospects for the antithrombotic effects of Psoraleae Fructus and the potential application of 4--MB as lead compounds in the therapy of thrombosis by targeting 14-3-3ζ.