To study the expression and clinical significance of cell division cycle factor 5 (CDC5L), DExD/H-box helicase (DDX21) and telomerase (hTERT) in colorectal cancer (CRC). Methods R language was used to analyze the expressions of CDC5L, DDX21 and hTERT in CRC cancer and adjacent tissues from the Human Cancer Genome Database. The protein expressions of CDC5L, DDX21 and hTERT in CRC carcinoma and adjacent tissues were detected by immunohistochemistry. The correlation of CDC5L, DDX21 and hTERT expression in CRC cancer tissues was analyzed by Pearson linear correlation analysis. The relationship between the expression of CDC5L, DDX21 and hTERT in cancer tissues and clinicopathological characteristics was statistically analyzed. Kaplan-Meier survival analysis (Log-rank test) were used to analyze the difference in survival prognosis of patients with different CDC5L, DDX21 and hTERT expression. Results Compared with adjacent tissues, the expressions of CDC5L, DDX21 and hTERT mRNA in CRC cancer tissues were significantly higher, and the difference was statistically significant (P＜0.001). The positive rates of CDC5L, DDX21 and hTERT in CRC cancer tissues were significantly higher than those in adjacent tissues (all P＜0.05). There was a significant positive correlation between CDC5L and DDX21 expression (rs=0.576, P＜0.001). DDX21 and hTERT expression were significantly positively correlated (s=0.521, P＜0.001). There was a significant positive correlation between CDC5L and hTERT expression were significantly positively correlated (rs=0.628, P＜0.001). There were significant differences in the expression of CDC5L, DDX21 and hTERT proteins in CRC cancer tissues with different tumor stages and lymph node metastasis (all P＜0.05). Kaplan-Meier survival curve (Log-rank test) analysis results showed that the 3-year overall survival rates of patients in the CDC5L positive expression group, DDX21 positive expression group, and hTERT positive expression group were significantly lower than those in the CDC5L negative expression group, DDX21 negative expression group, and hTERT negative expression group, respectively (all P＜0.05). Conclusion The expressions of CDC5L, DDX21 and hTERT are up-regulated in CRC cancer tissues. The expressions of CDC5L, DDX21 and hTERT are related to tumor stage and lymph node metastasis, and are new tumor markers for evaluating the prognosis of CRC