Objective To develop and verify an analytical method for the size of host cell residual DNA(HCD) fragments in MDCK cells based on magnetic bead method and quantitative real-time PCR(qPCR), so as to use it to evaluate the removal effect of HCD during the production process of pandemic influenza vaccine(H5N1).Methods The HCD of samples was extracted by magnetic bead method, and specific primers and probes were designed to establish qPCR analytical method that can quantify 84, 142, 204 and 504 bp DNA fragments, respectively. The established method was verified for the linear range,limit of quantitation(LOQ),precision, accuracy, robustness and specificity, and used to analyze the proportion of HCD fragments in the process samples of pandemic influenza vaccine(H5N1), and the HCD content was simultaneously detected.Results The linear ranges of 84, 142, 204 and 504 bp standards were 0. 003-300 pg/??L, showing a good linear relationship with Ct, and the coefficients of determination(R2) of the four standard curves were all greater than 0. 99. The LOQs of 84, 142 and204 bp were 3 fg/??L, and the LOQ of 504 bp was 1 fg/??L. The recovery rates of spiked samples at high, medium and low concentrations corresponding to 84, 142, 204 and 504 bp were all within the range of 50% to 150%. The CVs of precision,robustness and specificity verification were not greater than 40%. The proportion of large HCD fragments in the harvest solution of pandemic influenza vaccine(H5N1) was relatively high(≥ 204 bp fragments accounted for 81. 0%). After enzymatic hydrolysis and purification, the proportion of ≥ 204 bp fragments in the bulk solution was reduced to 8. 9%, and no ≥ 504 bp fragments were detected. At the same time, the total HCD content was reduced from 689. 9 ng/m L to 10. 2 ng/m L.ConclusionThe q PCR analytical method for HCD fragment size was developed with good precision, accuracy, robustness and specificity,which can be used for the analysis and detection of HCD fragments of pandemic influenza vaccine(H5N1) and the quality control in the production process.