Objective To develop a highly sensitive and accurate time-resolved immunofluorometric assay (TR-IFMA) for measurement of Dickkopf-1(DKK-1),as a novel serologic biomarker for lung cancer. Methods The study constructed a two-manoclonal-antibody "sandwich"-type assay and the sensitivity, within run CV and between run CV and accuracy were evaluated. Serum DKK-1 concentrations were measured by TR-IFMA in 120 healthy controls, in 72 benign lung disease patients, and in 212 lung cancer patients before surgery. The association between serum DKK-1 levels and clinicopathological features were evaluated. Results A standard curve for DKK-1 TR-IFMA had been developed with good sensitivity (0.08 μg/L). Both within run CV and between run CV were less than 6.5%. Accuracy studies, parallelism and precision data were determined and all found to be satisfactory. The validity of DKK-1 assay was confirmed by the good correlation between the results obtained by TR-IFMA and commercial ELISA (r=0.972, P=0.01). The serum levels of DKK-1 were higher in lung cancer patients 31.93(79.47-18.03) μg/L than in benign lung diseases 15.25(18.41-11.49) μg/L and in healthy controls 13. 90( 16. 91-11.02) μg/L DKK-1 levels were significant associated with the presence of distant metastases, as well as lymph node metastases and TNM stage, but not with patients' age, gender and tumor histology. At the cutoff of 22.63 μg/L, the diagnostic sensitivity, specificity, accuracy, positive predictive value and negative predictive value of the TR-IFMA for lung carcinoma were 68.4%, 92.2%, 82.1% ,90.6% and 72.5%. Diagnostic sensitivity and accuracy were higher for small cell carcinoma than for non-small cell carcinoma (70.7% vs 69.5% and 85.6% vs 80.7%, respectively). Conclusions A highly sensitive and reliable TR-IFMA for DKK-1 has been developed. The determination of serum DKK-1 levels may be useful for diagnosis and tumor staging of lung cancer.