A novel PCR-based mutagenesis method was reported, in which there is no need to purify megaprimers or design a special flanking primer. This method used one mutagenic primer and two sequencing primers (T_m≤58℃) as flanking primers. After first round PCR, 12.5 μl first PCR production was directly added into 50 μl second PCR system as template and megaprimer, and 10 rounds of asymmetrical PCR at high temperature of annealing (68℃) was to add in initiation of second PCR. This additional step greatly has increased the efficiency of mutagenesis via 600 bp or 800 bp long megaprimer. The results demonstrated that this method can achieve high fidelity, 97%～98% efficiency, high yield.