This thesis focused on the construction and characterization of Aspergillus flavus gene deletion mutants through gene complementation technique. The gene complementation cassette including the selection marker gene as well as the special epitope tag was successfully created through the combination of Expand Long PCR, Phusion PCR and Double-Joint PCR techniques, and subsequently introduced into the gene deletion strain via direct protoplast transformation. Successful integration of the complementation cassette was confirmed by diagnostic PCR and partial sequencing.