The invention discloses a liquid chromatography-mass spectrometry screening method for a multi-target antithrombotic substance based on a dried blood spot sample, and belongs to the field of drug screening. The method comprises the following steps of preparing a rehydrated dry blood spot sample, optionally selecting a plurality of substrates of key target enzymes involved in thrombosis processes,mixing a plurality of substrate standard solutions with a substance to be screened, adding a mixture into the rehydrated dry blood spot sample, treating a reaction solution, and carrying out liquid chromatography-mass spectrometry analysis to screen to obtain a substance with multi-target antithrombotic activity. The screening method of the invention has the advantages of simplicity and rapidness,high accuracy, high specificity, a large number of samples can be continuously analyzed, the antithrombotic substance capable of inhibiting one or more target enzymes at the same time can be screenedout at the same time according to the screening method, action targets of the screened active substance are clear, and a theoretical basis is laid for subsequent drug development. Reagents are easy to obtain, the operation automation degree is high, clinical popularization and promotion are easy, and the commercial value is high.