A method for determining the escherichia coli DNA content of a biological product is provided. The method includes subjecting the biological product to real-time fluorescence quantitative PCR detection by utilizing an escherichia coli specific primer group to determine the escherichia coli DNA content of the biological product. By utilizing the method in an embodiment, the escherichia coli DNA content of the biological product can be quantificationally analyzed, operation is simple and convenient, time consumed is short, sensitivity is high, and the lowest detection limit of the escherichia coli DNA content of the biological product can be 2.0 fg/microlitre. The method can provide reliable quality control bases for production of biotech medicines adopting escherichia coli as a host, and provides important supports for development and safe production of recombinant biotech medicines.