Objective To investigate the expression and clinical significance of cytokeratin 7 (CK7) and aspartic peptidase A (NapsinA) in pleural effusion of lung adenocarcinoma (LUAD). Methods A total of 86 patients with LUAD and pleural effusion who were admitted to Kaifeng Central Hospital from February 2023 to February 2025 were selected. Meanwhile, 40 samples of benign pleural effusion (pleural effusion caused by tuberculous pleurisy, heart failure, etc.) were collected. The expression of CK7 and NapsinA in pleural effusion cells was detected by immunohistochemical method. The relationship between CK7, NapsinA and clinicopathological features of patients with LUAD and pleural effusion was analyzed. Results The positive expression rates and scores of CK7 and NapsinA in pleural effusion of patients with LUAD were higher than those in benign pleural effusion (P<0.05). ROC curve showed that CK7 and NapsinA had vertain value in differential diagnosis of LUAD and other diseases (P<0.05). Among 86 patients with LUAD, CK7 expression and NapsinA expression were high in 37 cases (43.02%) and 41 cases (47.67%), respectively. No statistically significant differences were found in the high expression rates of CK7 and NapsinA when compared across patient gender, age, smoking status, T stage, and presence of extrathoracic metastasis (P>0.05). The high expression rate of CK7 in patients with epidermal growth factor receptor (EGFR) gene mutation was significantly higher than that in those without (P<0.05), but there was no statistically significant difference in the high expression rate of NapsinA between the two groups (P>0.05). Conclusion The positive expression rates of CK7 and NapsinA in pleural effusion of LUAD are high. Both have certain value in diagnosing LUAD and can serve as biological markers to distinguish benign and malignant pleural effusion. There is also a correlation between CK7 expression and EGFR gene mutation.