AbstractPleural mesothelioma is an aggressive tumor, commonly caused by exposure to asbestos. The prognosis of mesothelioma remains disappointing despite multimodal treatment. We reported previously that N‐ERC/mesothelin could be a useful biomarker for the early diagnosis of pleural mesothelioma and developed an enzyme‐linked immunosorbent assay (ELISA) system for its detection. However, the reproducibility of our previous 7–16 ELISA system has been revealed to be unsatisfactory. To measure N‐ERC/mesothelin more precisely, we developed a new 7–20 ELISA system. The subjects of this study were patients who were referred to our department with suspected pleural mesothelioma. The current study demonstrated that the newly established 7–20 ELISA system improved the sensitivity and specificity for diagnosing pleural mesothelioma compared with the previous system. Moreover, the 7–20 ELISA system showed better reproducibility and displayed the tendency of both higher sensitivity and higher specificity in plasma than in serum. Particularly for the epithelioid type, the area under the curve (AUC) and the diagnostic accuracy of N‐ERC/mesothelin were excellent; the AUC was 0.91, the sensitivity was 0.95, and the specificity was 0.76 in plasma. In conclusion, assessment of N‐ERC/mesothelin with our newly established 7–20 ELISA system is clinically useful for the precise diagnosis of pleural mesothelioma.