Abstract: OBJECTIVE To develop fast and accurate method for determining the residual DNA in PEGylated uricase (PEG-UHC) stock solution, and provide reference values for residual DNA quantification in other multiple-sites PEGylated protein drugs. METHODS The Quantitative PCR and DNA hybridization methods were used to determine the residual DNA based on "Exogenous residual DNA testing methods" in Chinese Pharmacopoeia 2020 edition Vol III. The detection limit, linearity, range, accuracy and precision of the quantitative PCR method were further evaluated. The residual DNA in three batches of PEG-UHC stock solution were determined by the above two methods. RESULTS The concentration ranges of DNA hybridization assay were 1×10^-4-0.1 ng·µL^-1, and there were no coloration upon the addition of PEG-UHC stock solution, indicating the quantities of residual DNA were below 10 ng per does in the three batches. The developed quantitative PCR had high detection limit up to 1×10^-5 ng·µL^-1 of DNA with good linearity(R²=0.999) in the concentration range of 1×10^-4-100 ng·µL^-1. The recoveries by adding different concentrations of standard DNA to PEG-UHC solution were 92.57%-114.17%, indicating no interference with PEG-UHC. The residual DNA of three batches of PEG-UHC stock solution was 0.143, 0.187, 0.154 ng per dose, respectively. All the contents met the National Medical Products Adiministration requirements for residual DNA. CONCLUSION The DNA hybridization assay is traditional qualitative methods, suffering from drawbacks of long time-consuming and low accuracy. The quantitative PCR assay has advantages of simple operation, high sensitivity, and can be used for quantitative analysis of residual DNA in multiple-sites PEGylated protein drugs.