To investigate the value of cytological smear and it combined with cell block immunohistochemistry in endoscopic ultrasonography guided fine needle aspiration biopsy (EUS-FNA). Methods 60 cases of patients underwent endoscopic ultrasonography guided fine needle aspiration biopsy due to occupying disease were collected. The diagnostic results of cytological smear and it combined with cell block immunohistochemistry were compared with results of surgical pathology or follow-up. Results The diagnostic sensitivity, specificity, accuracy of cytopathology for EUS-FNA were 78.6% (44/56), 50.0% (2/4), 76.7% (46/60) respectively. 19 patients underwent immunohistochemistry staining of cell block and cytopathology. The positive rate of identify tumor occupancy was 94.7% (18/19) and the accuracy was 94.7% (18/19). There was no significant difference in the detection rate of tumor occupancy in those 19 patients (P = 0.250). However, cytological smear combined with cell block immunohistochemistry is better than the method of cytological smear alone in distinguish pathological type from tumor. Conclusion There is no difference between cytological smear and its combined with cell block immunohistochemistry in the detection rate of endoscopic ultrasonography guided fine needle aspiration biopsy. Cytological smear combined with cell block immunohistochemistry is more advantageous in distinguish pathological type from tumor, and it can guide the follow-up treatment.