In order to improve the affinity of anti-CD3 ScFv, one site-directed mutagenesis method using “megaprimer” PCR was designed. Adopting the mutagenic primer in the first round of PCR, mutation DNA fragments of 180bp and 160 bp were amplified, respectively. Using these fragments as “megaprimers” in the second round of PCR, specific full-length DNA by changing the PCR program and adjusting the concentration of the primer and template were obtained. The sequencing results showed that the mutant pool of anti-CD3 ScFv had been yielded successfully.