The objective of this study was to explore the expression and clinical significance of Notch signaling genes in colorectal cancer. Colorectal cancer samples were prospectively collected from patients post-surgery at the 3rd Affiliated Hospital, Nanjing University of Traditional Chinese Medicine. Immunohistochemistry of tissue arrays was used to analyze the samples and genes involved in the Notch signaling pathway. Microsatellite instability (MSI) was detected by fluorescence multiplex polymerase chain reaction. A total of 146 colorectal cancer samples were collected, including 84 men (57.7%) and 62 women (42.5%). The average age of the study population was 60.8 ± 10.5 years. Notch1 and Notch2 gene expression correlated with tumor pathology type and degree of differentiation. In addition, Jagged 1 (JAG1) and hairy enhancer of split 1 gene expression correlated with the degree of tumor differentiation. Delta-like 1 gene expression varied significantly with tumor location. There was a significant difference between gene expression and MSI. Of the 138 patients, 134 (91.8%) participated in on-site visits, and the average follow-up time was 42.3 ± 13.3 months. During this period, 86 patients (71.6%) were tumor-free. At 1 year post-surgery, 93% of patients were alive, 74% of patients lived for 3 years, and 67% of patients lived for 5 years. The log-rank test was used to perform univariate analysis, and the COX proportional hazards model was used for the multivariate analysis. Based on these analyses, tumor prognosis correlated with the TNM stage, pathological type, microsatellite status as well as Notch2 and JAG1 gene expression. Patients expressing high levels of Notch2 and JAG1 presented with a significantly better prognosis compared to patients expressing negative or weak levels of Notch2 and JAG1. The expression levels of genes associated with the Notch signaling pathway correlated with tumor pathology and the degree of differentiation. In addition, Notch2 and JAG1 expression levels correlated with survival; however, the underlying mechanism for these correlations remains unclear.