AIM: To establish a quantitative measurement method of ECV-304 cell migration in a scratch wound model in vitro. METHODS: The method of ECV-304 cell scratch wound model was improved. The ECV-304 cell migration was measured quantitatively using a computer-assisted video microscopic method with image-analysis system. The effects of heparin and thrombospondin-1 on ECV-304 cell migration were observed in the scratch wound model in vitro. RESULTS: The average width and length of cell scratch wound was (0.95?0.08) mm and (51.20?3.40) mm respectively in 120 experiments. The peak of cell migration reached about 24 h after culture. The stimulated effect of heparin and inhibited effect of thrombospondin-1(TSP-1) on cell migration were observed. CONCLUSION: A quantitative measurement method of ECV-304 cell migration in vitro is established. The stimulated and inhibited effects on ECV-304 cell migration were proven respectively using this method.