Abstract: Hydrophobins are small amphiphilic proteins ubiquitously present in filamentous fungi, where they play essential roles in fungal growth, spore development, and host infection. To investigate the molecular characteristics and potential functions in Aspergillus flavus, seven putative hydrophobins were identified in the reference strain NRRL3357 and analyzed using integrated genomic and bioinformatics approaches. The coding sequences were retrieved from the NCBI database, while signal peptides, transmembrane regions, and GPI-anchor sites were predicted using SignalP 6.0, TMHMM, and PredGPI. The physicochemical properties of each protein, including molecular weight, theoretical isoelectric point, instability index, and hydrophobicity, were evaluated using the ExPASy ProtParam tool. Protein secondary structures were predicted with SOPMA, and three-dimensional models were constructed via homology modeling using SWISS-MODEL. Potential phosphorylation and glycosylation sites were identified using NetPhos 3.1 and NetNGlyc 1.0 to infer post-translational modification patterns. The results demonstrated that three class I hydrophobins (gene number AFLA_060780, AFLA_014260, AFLA_098380) possess complete conserved domains and exhibit high phosphorylation potential, suggesting their involvement in conidial surface hydrophobicity, spore attachment, and host recognition. Conversely, four intermediate-type hydrophobins (gene number AFLA_094600, AFLA_131460, AFLA_063080, AFLA_064900) displayed partial domain loss and diversified sequence features, indicating functional differentiation during environmental adaptation or infection. The predicted molecular weights ranged from 6.46 to 27.83 kDa, and the isoelectric points varied between 3.88 and 8.97. Secondary structure analysis revealed a predominance of random coils (45.00%-66.67%), reflecting significant molecular flexibility that may facilitate membrane assembly and surface film formation. Overall, this study provides a systematic characterization of hydrophobins in A. flavus, offering a theoretical foundation for understanding their biological roles in fungal development and infection, as well as identifying potential targets for mitigating crop contamination and aflatoxin risk.