In this study, we examined keratinase activity in twelve fungal isolates from various poultry markets, with isolate No.3 displaying the highest activity at 62.15 U/ml. Molecular analysis confirmed isolate 3 as Aspergillus flavus AFK3, a sequence deposited in the NCBI database. Sequential mutagenesis, involving ultraviolet (UV) radiation, ethyl methane sulfonate (EMS), and sodium azide (SA), led to the creation of the UV-33 mutant strain, which showed the most robust keratinase activity at 115.7 U/ml. Through Response Surface Methodology (RSM), we perfected culture conditions for the UV-33 mutant, achieving the highest keratinase activity at 133.73 U/ml under specific conditions of 1.5% malt extract, 1.5% fructose, pH 7, and 72 hours of incubation. A genetic diversity analysis compared A. flavus strain AFK3 with three mutants using inter-simple sequence repeat polymerase chain reaction (ISSR-PCR). The wild type AFK3 displayed the lowest band counts (28 bands with nonunique), while mutants UV-17, UV-33, and UV-24 had higher total and polymorphic bands, with UV-33 and UV-24 having 40 and 36 bands, respectively. Based on conserved regions, both A. flavus AFK3 and mutant A. flavus UV-33 were found to encode the subtilisin gene (SUB). The gene had an open reading frame of 2055 bp, which encoded a protein consisting of 684 amino acids. The 3D structure model was validated using Ramachandran's plot, indicating that residues were in the most favoured region and molecular docking studies revealed interactions with high-affinity scores for the template A. flavus strain CA14, wild type A. flavus AFK3, and mutant A. flavus UV-33 subtilisin, respectively.