Survivin and its splice variants DEx3 and 2B are involved in pathogenesis of numerous types of cancer. Proliferating cell nuclear antigen (PCNA) level correlates with cellular proliferation. The present study aimed to analyze the potential utility of survivin and its splice variants DEx3 and 2B as biomarkers for thyroid cancer. PCNA, survivin and its splice variants DEx3 and 2B expressions were analyzed in 22 tissue samples (15 thyroid cancers and 7 benign lesions) by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry (IHC). There was significantly higher staining for survivin (P=0.019), survivin DEx3 (P=0.001), survivin 2B (P=0.0149) and PCNA (P=0.0237) in thyroid malignant tumors when compared with benign lesions. The receiver operating characteristics curve analysis has shown that the cut-off points of survivin IHC expression >2 [sensitivity 46.7%; specificity 100%; area under curve (AUC) 0.810; P=0.0005] and survivin DEx3 IHC expression >0 (sensitivity 86.7%; specificity 100%; AUC 0.933; P1 (sensitivity 93.3%; specificity 71.4%; AUC 0.790; P=0.0243) and survivin 2B >2 (sensitivity 46.7%; specificity 100%; AUC 0.824; P=0.0002) were the best predictors of thyroid cancer. In conclusion, the present study exhibited that survivin DEx3 expression has high specificity and sensitivity for discrimination between benign thyroid lesions and cancers. Survivin DEx3 may be considered a biological marker of thyroid malignancy and therefore applied in clinical practice.