Abstract: A new method of real-time PCR was built to detect genetically modified microoganisms in microbioloogy derived enzyme. The primer and the MGB probe were desgined on the sequence of the alcohol oxidase-1 (AOX1) promoter. The experimental result showed that the sensitivity of detction was 1pg Pichia pastoris DNA, and could accurately analyze the residue status of genetically modified microorganism in microbiology derived enzyme from different separation stage. This practical method could be used to monitor separation of transgenic mocrobiology in microbiology dereirved enzyme, and provide reference for the detction of genetically modified microorganism related stdudies.