Objective To evaluate the roles of serum lactate dehydrogenase (LDH), pleural effusion adenosine deaminase (ADA) and their ratio in diagnosing malignant pleural effusion (MPE). Methods A retrospective analysis of 195 hospitalized patients with exudative pleural effusion from Nov. 2019 to Aug. 2022 was conducted. The patients were divided into 2 groups: MPE group (n＝103) and benign pleural effusion (BPE) group (n＝92, including 59 cases with tuberculous pleural effusion and 33 cases with bacterial pleural effusion). LDH levels in the peripheral blood samples and ADA levels in the pleural effusion samples were measured and compared in all enrolled subjects, and serum LDH/pleural effusion ADA ratios were calculated. The diagnostic efficacy of serum LDH, pleural effusion ADA and their ratio for MPE was evaluated by receiver operating characteristic curves. Results Patients in the MPE group had higher serum LDH levels and serum LDH/pleural effusion ADA ratios than those in the BPE group (215.00 ［185.00, 269.00］ U/L vs 175.00 ［155.25, 207.75］ U/L, 29.17 ［21.57, 44.83］ vs 6.96［5.05, 11.48］; both P＜0.001). Pleural effusion ADA levels of patients in the MPE group were lower than those in the BPE group (8.00 ［6.00, 10.00］ U/L vs 26.50 ［14.00, 36.00］ U/L, P＜0.001). The combined test of serum LDH, pleural effusion ADA and their ratio had high sensitivity and positive predictive values, among which the sensitivity and positive predictive value of serum LDH combined with serum LDH/pleural effusion ADA ratio were the highest for the diagnosis of MPE, reaching 0.95 and 0.94, respectively. Conclusion Serum LDH, pleural effusion ADA and their ratio have high diagnostic efficacy for MPE. The combined test can maintain the sensitivity and increase the positive predictive value, and serum LDH combined with serum LDH/pleural effusion ADA ratio is the best, which can distinguish benign and malignant pleural effusion earlier and has reference value for clinical decision making.