Only one bifunctional metal-chelator was used to prepare immunogen and coating antigen in all of the previous researches. However, the antibody-specific recognition to the spacer arm of the bifunctional metal-chelator might lower the specificity of heavy metal ions immunoassay. Two different bifunctional metal-chelators were adopted to prepare the immunogen and coating antigen respectively in our study to avoid this problem. The conjugates of keyhole limpet hemocyanin (KLH) and -SCN-Bz-DTPA-Pb were used as immunogen, whereas the conjugates of bovine serum albumin (BSA) and -NH -Bn-DTPA-Pb were used as coating antigen. Polyclonal antibodies specific to DTPA-Pb chelates were obtained from rabbits. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was adopted to detect Pb ion solutions prepared by Pb standard solution and ultrapure water. In the mixing microplate, DTPA and Pb ions formed chelates and combined with specific antibodies. After incubation, the DTPA-Pb and the antibodies complex were added into the wells of the reaction microplate. The reaction microplate was coated by the conjugates of BSA and -NH -DTPA-Pb, which competed for the specific antibodies. The result signals presented a good sigmoid curve when the Pb concentration ranges from 0.01 to 100 mg·L . The IC50 of the indirect competitive ELISA is 0.23±0.04 mg·L Pb ion. The cross-reaction with Cd , Cu , Fe , Mn , Zn , and other divalent ions were less than 5%.