Abstract: Objective To explore the accuracy of Ventana IHC to detect EML4-ALK in non-small cell lung cancer. Methods Total of 281 formalin-fixed paraffin-embedded samples (259 adenocarcinoma, 17 squamous carcinoma, 5 gland-scale cancer) diagnosed as NSCLC in Chinese PLA General Hospital from June 2014 to July 2016 were included in this study, and the EML4-ALK fusion gene of whom were detected by PCR and Ventana IHC, respectively. The PCR method was regarded as the golden standard, the positive rate between the two methods were compared and the sensitivity, specificity, positive predictive value and negative predictive value of Ventana IHC were calculated. Results The positive rates of PCR and Ventana IHC were 10.0% and 12.8%, respectively with no significant difference between the two methods (P=0.057). The sensitivity, specificity, positive predictive value and negative predictive value of Ventana IHC were 89.3%, 95.7%, 89.3% and 95.7%, respectively. Conclusion The Ventana IHC is efficient in detection of EML4-ALK fusion gene of NSCLC with favorable sensitivity, specificity, positive predictive value and negative predictive value, and it is economical, simple and convenient to be performed.