Background: The aim was to evaluate the diagnostic value of CYFRA21-1, Scc-Ag and telomerase activity in serum and pleural effusion in patients with squamous-cell lung cancer. CYFRA21-1, Scc-Ag and telomerase activity were measured using electrochemiluminescent, microparticle enzyme immunoassay and telomeric-repeat amplification protocol. Results: We evaluated 302 patients diagnosed with squamous-cell lung cancer and 153 patients diagnosed with benign lung diseases. All examined tumor markers were higher in pleural effusion than serum (p<0.05). The areas under receiver operating characteristic curve of CYFRA21-1 and Scc-Ag were higher in pleural effusion than serum. Optimizations for sensitivity, specificity and specificity in pleural effusion for CYFRA21-1 + Scc-Ag: 88.24, 97.17 and 92.79%; telomerase activity + Scc-Ag: 90.2, 95.28 and 92.8%; CYFRA21-1,Scc-Ag + telomerase activity: 91.18, 96.86 and 94.64%; respectively. Optimizations for sensitivity, specificity and specificity in serum for CYFRA21-1 + telomerase activity: 81.37, 94.34 and 87.98%; CYFRA21-1 + Scc-Ag and telomerase activity: 87.25, 93.08 and 90.8%; respectively. Conclusion: The combined detection of tumor markers in pleural effusion showed higher sensitivity, better accuracy and higher clinical value than serum and single detection. However, the cost effectiveness of these methods should be considered in clinical practice.