To develop an efficient gene-targeting platform in an excellent itaconic acid producing strain Aspergillus terreus CICC40205. The frequency of homologous recombination was improved by deleting the ku80 gene. A nutritional transformation system based on the bidirectionally selectable marker, pyrG (An) , was established in the ku80-/pyrG-double mutant which is convenient for following marker rescue. The modified Cre/loxP recombination system was applied for the excision of the pyrG (An) marker by directly introducing Cre recombinase into the protoplasts. This gene-targeting system is an efficient platform for sequential and multiple genetic modifications in A. terreus and is conducive to study biosynthesis mechanisms and to improve the production ability of itaconic acid and other products.