Bacterial fibrinolytic enzymes have an important role in thrombolytic therapy due to their ability to dissolve fibrin. Therefore, purification, characterization and activity determination are of prime importance for bacterial fibrinolytic enzymes. In the current study, marine Bacillus amyloliquefaciens was first screened from Sipunculus nudus living in the Beibu Gulf of China and denoted as Guangxi University-1 (GXU-1). Then, an extracellular fibrinolytic enzyme (FEB-1) was purified from GXU-1 using ammonium sulfate precipitation, hydrophobic chromatography and gel filtration chromatography. The specific activity of FEB-1 was determined to be as high as 6789.74 U/mg. The relative molecular weight of FEB-1 was measured as 30 kDa through SDS‒PAGE. The optimum in vitro fibrinolytic activity of FEB-1 was identified at 37 °C and pH = 8. Furthermore, the activity of FEB-1 can be well preserved at 20–45 °C and pH = 6.0 to 9.0. The combination analysis of SDS‒PAGE and the molecular docking calculation revealed that FEB-1 can cleave more Aα- and Bβ-chains of fibrinogen than γ-chain. It is noteworthy that FEB-1 is comparatively stable under human-body environmental conditions, indicating its potential application in thrombosis therapy.