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The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system is a powerful genome editing tool that has been successfully established in some filamentous fungi due to its high flexibility and efficiency. However, the potential toxicity of Cas9 restricts the further popularization and application of this system to some degree. The AMA1 element is a self-replicator derived from Aspergillus nidulans, and its derived vectors can be readily lost without selection. In this study, we eliminated Cas9 toxicity to Fusarium venenatum TB01 based on 100% AMA1-based Cas9 expression vector loss. Meanwhile, two available endogenous Pol III promoters (FvU6374 and Fv5SrRNA) used for sgRNA expression of the CRISPR/Cas9 system were excavated. Compared to FvU6374 (40–50%), Fv5SrRNA exhibited higher single-gene editing efficiency (> 85%), and the efficiency of simultaneous editing of the two genes using Fv5SrRNA was over 75%. Based on this system, a butanediol dehydrogenase encoding gene FvBDH was deleted, and the ethanol yield in variants increased by 52% compared with that of the wild-type. The highly efficient CRISPR/Cas9 system developed here lays the technical foundation for advancing the development of F. venenatum TB01 through metabolic engineering, and the obtained FvBDH gene–edited variants have the potential to simultaneously produce mycoprotein and ethanol by further gene modification and fermentation process optimization in the future. Key points • Cas9 toxicity disappeared and DNA-free gene-edited strains obtained after vector loss • Promoter Fv5SrRNA conferred TB01 higher gene editing efficiency than FvU6374 •Deletion of the FvBDH gene resulted in a 52% increase in ethanol yield [ABSTRACT FROM AUTHOR]