In this study, a planar benzothiazole Schiff base derivative ZX1 was designed and synthesized to selectively precipitate silver ions, in conjunction with the immunoassay detection of Hg2+ ions. The silver chelation process was highly effective in the pH range of 7–10, with a silver ion precipitation ratio of 99.9 %. The coordination behavior of ZX1 with Ag+ was studied using a series of spectroscopic measurements, topography analysis and DFT calculation. The shortest bond distances were 2.251 and 2.254 Å between nitrogen atom of thiazole and Ag+. Weak interactions were analyzed using the interaction region indicator (IRI) method and VMD. The immunoassay was developed through an anti-Hg2+/Ag+ monoclonal antibody, which showed similar sensitivities for Ag+ and Hg2+. The selectivity and anti-interference ability of the Schiff base ligands were tested for some hard metal ions and several anions. With co-existence of interference ion Ag+, Hg2+ was detected in rice matrix solution with 77 %∼123.5 % recovery by addition of ZX1 as the masking reagent and 90.3 %∼115 % for wheat matrix. This research provides a promising immunoassay strategy for simultaneous detection of silver ions and mercury ions in food matrix by utilization of Schiff base ligands. This novel Schiff base ligand was then used as the pre-treatment reagent to remove silver ions within 5 min for detection of Hg2+ or detection of both Ag+ and Hg2+ based on a novel bi-specific heavy metal antibody.