Objective To prepare the rabbit polyclonal antibodies against sixteen stains of birds，and to label the secondary antibodies with horseradish peroxidase for developing the serological test system of birds． Methods IgYs of birds were purified by water diluted，combining precipitation with ammonium sulfate，or affinity chromatography and precipitation with ammonium sulfate，or precipitation with ammonium sulfate and cutting the SDS-PAGE gel slices that contain the IgY bands． Rabbit anti-sera were prepared by repeatedly immunized rabbits with purified IgYs． The antibodies against bird IgY in rabbit sera were detected by agarose double immunodiffusion and purified using Protein A affinity chromatography． The products were labeled using HRP by periodate oxidation method． And the activities of the labeled rabbit polyclonal antibodies against sixteen strains of birds were detected by ELISA． Western blots were performed to investigate the specificity of IgG-HRP against the IgY of birds． Results The IgYs from the strains of birds were purified，including greylag goose，great cormorant，ostrich，little grebe，pigeon，rich bird，goose，peacock，quail，ladyhood-chicken，heron， night-heron，red-crested pochard，tern，black-winged stilt，parrots，common shelduck． The titers of sixteen rabbit antisera were up to 1 ∶ 32 examined by agarose double immunodiffusion． Sixteen kinds of purified rabbit anti-bird secondary antibodies were labeled with HRP and their activities were up to 1∶ 800 － 80000 examined by ELISA． The specific activities of the sixteen rabbit anti-bird IgGs-HRP were indicated with western blot． Conclusions Rabbit polyclonal antibodiesagainst sixteen birds labeled with HRP were prepared successfully; It was an useful tool for developing the serological test system for birds．