There is a lack of study concerning expression of Topoisomerase IIα (Topo IIα) and long-term results in colorectal cancer patients. We aimed to investigate the relationship between expression of Topo IIα and clinicopathological parameters including overall survival in colorectal cancer. Paraffin-fixed specimens from a large prospective cohort of colorectal cancer patients who had been followed up for 4 years were assayed immunohistochemically. Of 490 colorectal cancer patients accessible for Topo IIα expression, expression of Topo IIα was scored as (-) in 4 (0.8%) patients, (+) in 41 (8.4%) patients, (++) in 396 (80.8%) patients, and (+++) in 49 (10.0%) patients. Overexpression of Topo IIα was found to be related with lower T stage (p = 0.042), lower N stage (p = 0.038), and a lower incidence of recurrence with nearly significance (p = 0.053). Kaplan-Meier analyses showed that overexpression of Topo IIα was related with prolonged overall survival (p = 0.022) and disease-free survival (p = 0.036). Multivariate analyses showed that elevated serum CEA (p < 0.001), elevated serum CA199 (p = 0.002), poor differentiation (p = 0.001), advanced Dukes stage (p < 0.001), and lower expression of Topo IIα (p = 0.017) were independent predictive factors for poor prognosis. Topo IIα expression is a valuable prognostic indicator for colorectal cancer and would be useful in treatment selection for early colorectal cancer and malignant colorectal polyps resected under endoscopy, especially when it is used in combination with serum CEA, CA199, and differentiation.