Objective To optimize the experimental conditions for the determination of porcine endogenous retrovirus (PERV) copy number in the porcine genome using a droplet digital polymerase chain reaction ( ddPCR) technique. Methods TaqMan probe duplex-ddPCR was used to determine the copy number of PERV, glyceraldehyde 3-phosphate dehydrogenase ( GAPDH) and transferrin receptor protein 1 ( TFRC) were adopted as the internal reference genes. To determine the optimal annealing temperature and PCR cycle number, we compared multiple conditions. In addition, the suitable range of template amount was estimated by examining the serially diluted plasmid and the pig cell genome. Genomic DNA isolated from pig cell lines or Bama minipig tissue was used to determine the copy number of PERV using ddPCR-and quantitative PCR-based method, which demonstrated the repeatability of different method. Results The optimal annealing temperature for the ddPCR-based method to measure PERV copy number was 59. 9℃ , and the optimal number of PCR cycles was 50. The suitable template genome concentration ranged from 1. 25 to 7. 5 ng. The variation of coefficients for PERV copy number in the porcine genome ranged from 0. 44% to 8. 29%. Conclusions A method for analyzing the PERV copy number based on the ddPCR technique was established, which improved the accuracy and repeatability compared with a traditional quantitative PCR-based detection approach. This method provides a sensitive approach and is a reliable basis for the determination of PERV copy number in donor animal genomes for xenotransplantation studies.