Natural autoantibody is a key component for immune surveillance function. Regulatory T (Treg) cells play indispensable roles in promoting tumorigenesis via immune escape mechanisms. Both CD25 and FOXP3 are specific markers for Treg cells and their natural autoantibodies may be involved in anticancer activities. This work was designed to develop an in-house enzyme-linked immunosorbent assay (ELISA) to examine plasma natural IgG against CD25 and FOXP3 in non-small cell lung cancer (NSCLC). Compared with control subjects, NSCLC patients had significantly higher levels of plasma IgG for CD25a (Z= -8.05, P<0.001) and FOXP3 (Z= -4.17, P<0.001), lower levels for CD25b (Z= -3.58, P<0.001), and a trend toward lower levels for CD25c (Z= -1.70, P=0.09). Interestingly, the anti-CD25b IgG assay had a sensitivity of 25.0% against a specificity of 95.0% in an early stage patients (T1N0M0) who showed the lowest anti-CD25b IgG levels among 4 subgroups classified based on staging information. Kaplan-Meier survival analysis showed that patients with high anti-FOXP3 IgG levels had shorter survival than those with low anti-FOXP3 IgG levels (χ2=3.75, P=0.05). In conclusion, anti-CD25b IgG may be a promising biomarker in terms of screening individuals at high risk of lung cancer.