Background: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive technique for cytological and histological diagnosis. The objective of this study was to explore the role of cytological diagnosis in EBUS-TBNAs. Methods: Eight hundred and thirteen consecutive cases performed EBUS-TBNA with both cytological and histological diagnoses were retrospectively reviewed. All patients were followed up for clinical data. Results: Before immunohistochemical examination, the cytological sensitivity, specificity, and diagnostic accuracy of EBUS-TBNAs were 92.9% (421/453), 98.9% (348/352), 95.5% (769/805), respectively. After immunohistochemical examination, the sensitivity, specificity, and diagnostic accuracy were 93.0% (423/455), 99.4% (348/350), 95.8% (771/805), respectively. The majority of false-negative were cases whose cytological diagnosis was "atypical" or the cytological diagnosis suggested "inadequate." "Neoplastic" were also prone to false-negative cytology. The diagnostic accordance rate of cytological subtyping was 90.3% for squamous-cell carcinoma, 99.2% for adenocarcinoma, and 98.1% for small-cell carcinoma before immunohistochemical examination, and became 85.9%, 98.5%, and 98.2% after immunohistochemical examination, respectively. Conclusion: Cytological diagnosis in EBUS-TBNAs had a good sensitivity and high specificity. The sensitivity and specificity of cytological diagnosis were proved to be higher after the immunohistochemical examination. At the same time, cytology had high accordance rate in subtype diagnosis. False-negative results occurred more commonly in cases whose cytological diagnosis was "atypical" or the cytological diagnosis suggested "inadequate" or the corresponding histological diagnosis was "Neoplastic.