Abstract: In order to establish a rapid, sensitive and specific detection method for waterfowl circovirus, three specific primers were designed according to the Rep gene sequences of Duck circovirus（DuCV） and Goose circovirus（GoCV） published in GenBank. Through optimization of amplification conditions and specificity and sensitivity tests, 40 diseased samples suspected of waterfowl circovirus infection were detected by the established nested PCR detection method for waterfowl circovirus. The results showed that the nested PCR method of waterfowl circovirus could successfully amplify the target fragments of 719 bp and 543 bp. The annealing temperature in the optimized first and second rounds of PCR was 50 ℃ and 56 ℃, respectively; the amount of primers added was 0.6 μL; the template dilution ratio of the second round of PCR was 1×10~3 times. The optimized method had good specificity for the detection of waterfowl circovirus; there was no cross-reaction with Duck hepatitis A virus type 1（DHAV-1）, Duck hepatitis A virus type 3（DHAV-3）, Duck Tembusu virus（DTMUV）, H5 N6 subtype Influenza A virus（AIV）, H9 N2 AIV, Newcastle disease virus（NDV）, Anatid alphaherpesvirus 1（DEV）, Goose parvovirus（GPV） and Goose astrovirus（GoAstV）. The detection limit of this method was 4.04×10~2 copies/μL, which were 1×10~4 and 1×10~3 times the detection limit of the first and second rounds of PCR respectively. By using this method to detect diseased samples, the positive rate of GoCV was 60%（12/20）, and the positive rate of DuCV was 40%（8/20）. The results suggested that the nested PCR detection method established in the experiment could be used for the detection of waterfowl circovirus.