Abstract: OBJECTIVE To establish a Real-time PCR method for quantitative detection of the Pichia pastoris DNA residues in ornithine aspartate. METHODS Genomic DNA of Pichia pastoris was extracted by using host cell residual DNA sample pretreatment kit based on magnetic beads. The reaction system and condition for Real-time PCR were determined, and a standard curve was plotted based on residual DNA kit for detection of Pichia pastoris by fluorescent probe. The Real-time PCR method was verified for accuracy and precision. RESULTS The calibration curve was linear in the range of 0.03-300 pg·μL^-1, with r>0.99, and the limit of quantitation was 0.03 pg·μL^-1. The residual Pichia pastoris DNA content in ornithine aspartate bulk of conbercept was far less than 10 ng per dose. CONCLUSION The method can be used for the quantitative determination of Pichia pastoris DNA residues in ornithine aspartate.