Background and objective Telomerase and CYFRA21-1 may be positively expressed in malignant pleural effusion, but the sensitivity and specificity of single tumor marker were low. The aim of this study is to investigate the diagnostic value of combining determination of telomerase activity and CYFRA21-1 levels in differentiating benign from malignant pleural effusion caused by lung cancer. Methods 80 patients with malignant and 50 patients with benign pleural effusion were enrolled into this study. The telomerase activity in pleural effusion was tested by means of telomeric repeat amplification protocal-PCR-ELISA (TRAP-PCR-ELISA) and CYFRA21-1 levels were tested by the EIA method. All the results were analyzed by the statistical method. Results The levels of telomerase and CYFRA21-1 in malignant pleural effusion was significantly higher than that in benign one (t =17.252 and t =13.951, P<0.001). The sensitivity of telomerase activity testing for diagnosing malignant pleural effusion was 71.3%; the specificity was 86.0% and the overall accuracy was 76.9%. The sensitivity of CYFRA 21-1 testing was 60.0%, the specificity was 78.0% and the overall accuracy was 66.9%. The sensitivity of the combined testing was 90.0%, the specificity was 76.0% and the overall accuracy was 86.9%. The sensitivity and the overall accuracy of combined testing were higher than those of telomerase and CYFRA21-1 testing single(χ2=9.002 and χ2=19.201, P<0.01; χ2=4.389 and χ2=14.647, P<0.05). Conclusion The combined testing of telomerase with CYFRA21-1 can increase the sensitivity and overall accuracy of differential diagnosis of benign and malignant pleural effusion diagnosis.