The invention discloses antithrombotic heparin extracted from short necked clam and a preparation method and application of the antithrombotic heparin. The invention is characterized in that the structural formula of the heparin tetrasaccharide is as shown in (I), and the preparation method comprises the following steps: adding distilled water into short necked clam meat powder to dissolve a sample, regulating the pH value to 7.8-8.2, adding alkaline protease and papain to carryi out enzymolysis to obtain an enzymatic hydrolysate, carrying out enzyme deactivation, centrifuging, and taking a supernatant; transferring the supernatant into a balanced chromatographic column with macroporous ion exchange resin, taking distilled water as a solvent in the column, and combining and collecting elution components of a 1.5 M NaCl solution; concentrating the collected eluted component, adding ethanol, standing, centrifuging, taking a precipitate, alternately washing the precipitate with acetone and ethanol, dissolving the precipitate with water, centrifuging to remove insoluble substances, and repeating the operation for 3-4 times; and finally, dialyzing, and freeze-drying to obtain the antithrombotic heparin product extracted from short necked clam. The invention has the advantages of low bleeding side effect, mild anticoagulation effect and strong fibrinolysis effect.