SPLUNC1 (Short palate, lung and nasal epithelium clone1) protein is an abundant secretory product of epithelia present throughout the conducting airways. Although its function is still not fully known, most studies have focused on its defensive effect in the infection of human airways and its potential to serve as a molecular marker for lung cancer. In this study, we further evaluated the SPLUNC1 expression in patients with lung disease to explore its role in cancer or tuberculosis at the protein level. We generated a panel of antibodies by using protein from a eukaryotic expression system as the immunogen to mice. It was the panel of SPLUNC1 monoclonal antibodies that allowed us to comparatively determine SPLUNC1 protein in lung cancer and tuberculosis infection by detecting sera and pleural effusion other than airway surface. The results showed that the SPLUNC1 level was not significantly changed either from sera of lung cancer or control. There was a significant increase in pleural effusion from lung cancer when compared to tuberculosis. These results indicate that SPLUNC1 may be a useful marker for tracing lung cancer cells, based on its epithelial origin property in pleural effusion.