Objective To evaluate the potential of S100 calcium binding proteins S100A2 and S100A6 levels in serum as diagnostic markers for non-small cell lung cancer (NSCLC). Methods Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of S100A2 and S100A6 in 141 NSCLC patients and 150 healthy subjects. Results The average level of serum S100A2 in NSCLC group was (15.02±0.79) ng/ml, that in healthy control group was (11.18±0.64) ng/ml, and the difference was statistically significant (P=0.002). The average level of serum S100A6 in NSCLC group was (12 760±651.8) pg/ml, that in healthy control group was (8 434±408.2) pg/ml, and the difference was statistically significant (P<0.001). The serum levels of S100A2 and S100A6 in stageⅠ/ⅡNSCLC patients were higher than those in healthy controls (P<0.05), respectively. Receiver operating characteristic (ROC) curve analysis showed that S100A2 and S100A6 could distinguish NSCLC patients from healthy controls [area under the curve (AUC) were 0.646 and 0.668, respectively]. Meanwhile, these two proteins showed notable capabilities for distinguishing stage Ⅰ/ⅡNSCLC from healthy controls (AUC were 0.708 and 0.702, respectively). Conclusion Serum levels of S100A2 and S100A6 are significantly elevated in the early stage for NSCLC patients, which can be the potential biomarkers for the diagnosis of NSCLC.