Objective: To analysis the application value of cell block of pleural effusion in the diagnosis of advanced cancer and detection of EGFR/ALK/ROS1 in lung adenocarcinoma. Methods: The remaining cytological specimens after routine smear were collected to prepare pleural effusion cell block. HE staining was preformed. Different antibodies were selected according to different origins of tumor. We classify tumors of malignant pleural effusion combined with HE morphology and immunohistochemistry results. At the same time, the EGFR/ALK/ROS1 gene status was detected in cases with a clear diagnosis of lung adenocarcinoma. Results: In 79 patients, 60 cases were found cancer cells, 15 cases were proliferating mesothelial cells and 4 cases were atypical cells difficult to make an accurate judgment. Sixty malignant cases, 51 cases were adenocarcinoma, 4 cases small cell carcinoma, 4 cases squamous cell carcinoma, 1 case spindle cell sarcoma, and 1 case diffuse large B-cell lymphoma. According to immunohistochemistry and clinical history, 43 cases were lung adenocarcinoma, 2 cases breast cancer, 2 cases ovary carcinoma, 2 gastric adenocarcinoma, 1 colon cancer, and 1 liver cancer. Forty-three lung adenocarcinoma cases, the EGFR/ALK/ROS1 gene tests were detected in 22 patients. The results showed that the EGFR mutation rate was 54.5% (12/22), and the ALK and ROS1 gene fusion mutation rates were 9.1% and 4.5% (2/22 and 1/1), respectively. Conclusion: Pleural effusion cell blocks, combined with immunohistochemical staining can help the diagnosis of advanced tumors and provide assistance in the selection of individualized treatment options for patients with lung adenocarcinoma.