A novel artificial antigen (AA) for Ni2+ was prepared by designed route, which coupled diethylenetriamine-N,N,N,N',N"-pentaacetic dianhydride (DTPAA) with bovine serum albumin (BSA) and human serum albumin (HSA), and then chelated the DTPAA-BSA/HSA with Ni2+ The AA was characterized by bicinchoninic acid (BCA) protein assay, atomic absorption spectroscopy (AAS), ultraviolet (UV) spectrum, fluorescence spectrum, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 2,4,6-trinitro-benzenesulfonic acid assay (TNBS) The consistent results showed that the synthetic route different from the traditional method was effective, and the AA was synthesized successfully