Abstract: The present study aims to explore the prognostic role of Notch3 pathway in gastrointestinal cancers. The expression pattern of Notch3 was investigated using the Timer and GEPIA web-tool at pan-cancer level. Expression levels of Notch3 pathway-related genes were compared between tumor and normal tissues based on GSE31168 and GSE11417 datasets. Expressional correlation among Notch3 pathway-related genes was further explored using Spearman correlation test in these two datasets. The association between Notch3 expression and prognosis of patients with gastric cancer or colon cancer was analyzed using genomic DNA data and clinical information from TCGA-STAD and TCGA-COAD datasets respectively. A risk assessment model based on Notch3 pathway-related genes was constructed for the prognostic prediction for patients with STAD or COAD using multivariate Cox regression analysis. The results revealed the high expression of Notch3 in both gastric cancer and colon adenocarcinoma. Notch3 expression was positively and significantly correlated with the expression of DLL1, DLL3, HEY1, HEYL and FLT4 in STAD. Higher expression of Notch3, DLL1, DLL3, HEY1, HEYL and FLT4 predicted poorer prognosis in STAD. Similarly, Notch3 expression was positively and significantly correlated with the expression of DLL1, DLL3, HEYL and FLT4 in COAD. Higher expression of Notch3, DLL1, DLL3, HEYL and FLT4 predicted poorer prognosis in COAD. The risk assessment model based on Notch3 pathway-related genes performed better in prognosis predicting for gastric cancer than colon cancer. Higher expression of Notch3-related gene signature predicted poorer prognosis in gastrointestinal cancer, and provided a new idea for the underlying mechanism and prognosis analysis by further bioinformatics analysis.