Abstract: Objective: An auxotrophic strain with high efficiency of homologous recombination was developed in Aspergillus oryzae RIB40. Methods: The AopyrG mutant of A. oryzae RIB40 was firstly obtained using a homologous recombination technique and the 5-fluoroorotic acid (5-FOA) selection for the uridine/uracil auxotroph. To disrupt the ∆Aoku70 gene, the Aoku70 gene of AopyrG mutant was replaced with A. fumigatus pyrG gene. Then, under the screening effect of 5-FOA, the AfpyrG gene of the ∆Aoku70 strain was deleted via genome self-cyclization. To test the utilization of the resulting strain, the mCherry gene, a red fluorescent protein gene, was integrated with the histone H2B gene in the ∆Aoku70∆AopyrG strain. The subcellular localization of red fluorescent protein was detected by laser confocal microscope. Results: An auxotrophic mutant ∆Aoku70∆AopyrG with high homologous recombination efficiency was established via disruption of the A. oryzae RIB40 genes Aoku70 and AopyrG. The red fluorescence was observed in the nucleus of the fungal cell, indicating the utility of the ∆Aoku70∆AopyrG strain. Conclusion: The ∆Aoku70∆AopyrG strain can be used as a recipient strain with high-efficiency homologous recombination and uridine/uracil auxotroph for future gene modification in A. oryzae RIB40.