To identify potential biomarkers related with lung cancer metastasis, conditional media (CM) proteins collected from a primary non-small cell lung cancer (NSCLC) cell line NCI-H226 and its brain metastatic subline H226Br were analyzed by one-dimensional electrophoresis (1-D PAGE) and matrix-assisted laser desorption/time of flight mass spectrometry (MALDI-TOF-MS). Twelve biomarkers were identified, of which l-lactate dehydrogenase B (LDHB) chain was significantly up-regulated in the CM of H226Br cell and was further validated in 105 lung cancer, 93 non-lung cancer, 41 benign lung disease, as well as 65 healthy individuals sera using enzyme-linked immunosorbent assay (ELISA). It was found that the levels of LDHB were specifically elevated in NSCLC sera compared with other groups and were progressively increased with the clinical stage. At the cutoff point 0.260 (OD value) on the receiver operating characteristic (ROC) curve, LDHB could comparatively discriminate lung cancer from benign lung disease and healthy control groups with sensitivity 81%, specificity 70% and total accuracy 76%. These findings demonstrated that secretome could open up a possibility to find, identify, and characterize novel biomarkers related with invasion and metastasis.