The aim of this study was to investigate topoisomerase II alpha (TOP2α) overexpression and its association with clinicopathological features and prognosis in gastric cancer (GC) patients. All selected GC patients at Affiliated Hospital of Qinghai University and Cancer Hospital, Chinese Academy of Medical Sciences, between December 2009 and December 2011, had formalin-fixed and paraffin-embedded tumor tissues. The patients received a telephone follow-up or in-/outpatient review, and their clinicopathological features and prognoses were analyzed. Also, the relationship between TOP2α expression and postoperative chemotherapy in GC patients was estimated. The results of the study showed that TOP2α overexpression correlated with location of tumor, depth of invasion, and pTNM stage. Moreover, it was associated with lower 5-year overall survival (OS) in noncardia GC patients younger than 60 years, with multivariate analysis demonstrating that it was an independent prognostic factor for these patients. Univariate analysis and multivariate analysis showed that TOP2α overexpression was associated with worse 5-year OS in noncardia GC patients ≤ 60 years receiving postoperative chemotherapy. TOP2α overexpression exhibited associations with location of tumor, depth of invasion, pTNM stage, and postoperative chemotherapy, making it a potential target for early diagnosis of GC patients. In addition, TOP2α overexpression was shown to be a predictor of 5-year OS in both noncardia GC patients ≤ 60 years and noncardia GC patients ≤ 60 years and receiving postoperative chemotherapy.