Reliable and noninvasive biomarkers for the early diagnosis of non‐small‐cell lung cancer (NSCLC) are an unmet need. This study aimed to screen and validate potential urinary biomarkers for the early diagnosis of NSCLC. Using protein mass spectrometry, urinary MDH2 was found to be abundant both in patients with lung cancer and lung cancer model mice compared with controls. Urine samples obtained as retrospective and prospective cohorts including 1091 NSCLC patients and 736 healthy controls were measured using ELISA. Patients with stage I NSCLC had higher urinary MDH2 compared with healthy controls. The area under the receiver‐operating characteristic curve (AUC) for the urinary MDH2 was 0.7679 and 0.7234 in retrospective and prospective cohorts to distinguish stage I cases from controls. Urinary MDH2 levels correlated with gender and smoking history. MDH2 expression levels were elevated in lung cancer tissues. MDH2 knockdown using shRNA inhibited the proliferation of lung cancer cells. Our study demonstrated that urinary MDH2 concentration was higher in early‐stage NSCLC patients compared with that in controls and that MDH2 could serve as a potential biomarker for early detection of NSCLC. Malate dehydrogenase 2 was significantly elevated both in urine and in cancer tissues of NSCLC patients. The level of MDH2 in urine could serve as an assistant biomarker for the early diagnosis of NSCLC.