<p>The filamentous fungus <i>Aspergillus oryzae</i> is a critical microbial cell factory for organic acid production. However, its efficient utilization of complex carbon sources is significantly hindered by carbon catabolite repression (CCR) mediated by the transcription factor CreA. A comprehensive understanding of CreA’s DNA recognition mechanism is crucial for overcoming this limitation. This study aims to decipher the collaborative DNA-binding mechanism between the zinc finger domain (ZFD) and the C-terminal basic region of CreA. We engineered a truncated protein, ZFDB, containing both domains. Using fluorescence polarization assay, we demonstrate that the basic region dramatically enhances the DNA-binding affinity of the ZFD by approximately one order of magnitude through specific interactions with the 5’-flanking sequences of the core DNA motif. Predicted structural modeling reveals a novel synergistic mechanism: the ZFD occupies the major groove while the basic region forms an α-helix that traverses the phosphate backbone and penetrates the minor groove. Mutagenesis studies showed that enhancing DNA flexibility and reducing steric hindrance in the basic region significantly strengthened binding. Strikingly, we discovered that DNA binding is substantially enhanced at lower pH (from 7.4 to 4.5), leading us to propose a hypothesis for a pH-mediated CCR regulatory model. Our findings suggest the first evidence of a dual-groove, pH-sensitive DNA recognition mechanism in CreA, offering novel perspectives on CCR regulation and providing a potential framework for developing <i>A. oryzae</i> strains with enhanced carbon source adaptability for industrial applications.</p>