Objective To study the feasibility of establishing human 3D pancreatic cancer organoids with endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimen. Methods A total of 9 patients with suspected pancreatic masses were prospectively included in this study from June 2017 to January 2018. EUS-FNA was performed for initial diagnosis. The biopsy tissues were obtained from a COOK 22-gauge FNA needle for organoid establishment, and the growth status in vitro was observed. Results Nine specimens of pancreatic lesions obtained from EUS-FNA were enrolled. Successful establishment of organoids was achieved in 5 cases, which were all confirmed to be pancreatic cancer histopathologically. In the process of generating, the growth rate of organoids increased correspondingly. The pathological morphology of these organoids was similar to the corresponding pancreatic tissues in HE staining. Conclusion Pancreatic cancer organoids can be successfully created by means of EUS-FNA. Establishment of these organoids can potentially provide excellent models for patients with pancreatic cancer in guiding precision treatment.