Abstract: MYB-type transcription factors are involved in regulating biological growth and development, metabolism, and responses to external stresses. To investigate the function of MYB-type transcription factors in Aspergillus flavus (A. flavus), a deletion mutant of the transcription factor AflbasR-deficient strain was constructed through homologous recombination, and its effects on the growth, conidial production, aflatoxin B₁ (AFB₁) synthesis, stress tolerance, and pathogenicity of A. flavus were analyzed. The results showed that compared with the control strain, the ΔAflbasR strain exhibited slower growth, denser and larger conidial heads, and shorter and thicker conidial peduncle. The deletion of AflbasR gene significantly reduced the tolerance of A. flavus to the cell wall stress agent Congo Red and oxidative stress (H₂O₂). The biosynthesis of AFB₁ was significantly decreased, and the ability to infect peanut seeds was reduced. This study enriches the theory of aflatoxin biosynthesis regulation and provides potential targets for preventing A. flavus contamination.