The invention discloses low-immunogenicity lumbrukinase, a preparation method and application thereof, and belongs to the field of biological pharmacy. The method provided by the invention comprises the following steps of: first, extracting to obtain a lumbrukinase crude product from an earthworm, and then, passing the crude product through an ion exchange column to elute, purifying, colleting a component with fibrinolytic activity, freezing, drying, concentrating, separating a concentrated lumbrukinase solution by using a gel filtration chromatographic column, detecting the fibrinolytic activity by using an agarose-hemaleucin flat band method, collecting a second eluting peak with the fibrinolytic activity, wherein detected by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electropheresis), the molecular weight of the second eluting peak is 23500-27500 Da; and after the eluting peak is dialyzed, desalted and subjected to low-temperature freeze drying, carrying out molecular modification on the eluting peak by utilizing activated monomethoxy polyethyleneglycol and dextran to obtain the low-immunogenicity lumbrukinase. The lumbrukinase prepared by the invention has the characteristics of low immunogenicity, high enzyme activity and the like, and can be used for the research and the preparation of an antithrombotic drug and a drug for treating or preventing cardiovascular and cerebrovascular diseases.