Abstract： Objective： To investigate the effects of preparation process optimization of earthworm extract on its anticoagulation and amino acid content. Methods：By taking decocting time，alcohol settling ratio and drying method as influencing factors，and by using anticoagulation and amino acid content as evaluation indexes， the effects of preparation process optimization of earthworm extract on its anticoagulation and amino acid content were analyzed according to the design for the orthogonal L 9 (3 4 ) test. Results：Decocting time(A)，alcohol settling ratio(B) and drying method(C) had significant effects on the anticoagulation and amino acid content of the extract(P＜0.05)，and the order of influence was A＞B＞C. The longer the decocting time，the higher the alcohol settling ratio，the higher the anticoagulation and amino acid content of the extract. The extract obtained by freeze-dried method had better appearance and color，whose anticoagulation was the best，amino acid content highest，and water content the lowest. Taking anticoagulation and amino acid content as indexes，the optimal process was decocting for three hours，alcohol settling ratio 70%，and freeze-drying. The three batches of earthworm extract prepared by the optimal preparation process were loose，porous，light yellow and were without collapse in appearance， whose average water content was 2.81%，average anticoagulation 484 IU/mg and average total amino acid content 3.74%；the relative standard deviation of each index of the three batches was less than 5%，and the difference between the three batches was small. Amino acid content was positively correlated with anticoagulation(r=0.986，P=0.000). Conclusion：The preparation process of earthworm extract influences the anticoagulation by affecting the amino acid content. The optimal process is decocting for three hours， alcohol settling ratio 70%，and freeze-drying，so that the difference between batches of extract is small and the reproducibility is good.