This research was conducted to compare differences in colon cancer lymphatic vessel invasion (LVI) with D2-40 antibody labeling and regular HE staining, blood vessel invasion (BVI) with CD34 antibody labeling and HE staining and to assess the possibility of using D2-40-LVI/CD34-BVI in combination for predicting stage II colon cancer prognosis and guiding adjuvant chemotherapy.Anti-D2-40 and anti-CD34 antibodies were applied to tissue samples of 220 cases of stage II colon cancer to label lymphatic vessels and small blood vessels, respectively. LVI and BVI were assessed and multivariate COX regression analysis was performed for associations with colon cancer prognosis. Regular HE staining proved unable to differentiate lymphatic vessels from blood vessels, while D2-40 selectively labeled lymphatic endothelial cell cytosol and CD34 was widely expressed in large and small blood vessels of tumors as well as normal tissues. Compared to regular HE staining, D2-40-labeling for LVI and CD34-labeling for BVI significantly increased positive rate (22.3% vs 10.0% for LVI, and 19.1% vs 9.1% for BVI). Multivariate analysis indicated that TNM stage, pathology tissue type, post-surgery adjuvant chemotherapy, D2-40-LVI, and CD34-BVI were independent factors affecting whole group colon cancer prognosis, while HE staining-BVI, HE staining-LVI were not significantly related. When CD34-BVI/D2-40-LVI were used in combination for detection, the risk of death for patients with two or one positive results was 5.003 times that in the LVI(-)&BVI(-) group (95% CI 2.365 - 9.679). D2-40 antibody LVI labeling and CD34 antibody BVI labeling have higher specificity and accuracy than regular HE staining and can be used as molecular biological indicators for prognosis prediction and guidance of adjuvant chemotherapy for stage II colon cancer.