Laccase,　a　multicopper　oxidase,　is　well　known　for　its　industrial　potentials　to　remove　environmental　pollutants　due　to　its　low　substrate　specificity　to　oxidize　phenols　and　thus　catalytic　versatility.　Many　efforts　focused　on　the　metabolic　mechanism,　yet　to　decipher　the　structural　determinants　responsible　for　the　differentiation　between　substrates.　Aflatoxin　B1　(AFB1),　a　new　substrate　for　laccase,　is　a　mycotoxin　with　a　formidable　environmental　threat　to　public　health　and　food　safety.　In　the　present　study,　we　combined　biochemical,　in　silico　mutational　and　molecular-docking　data　to　gain　an　insight　to　the　function　of　key　residues　in　the　active　cavity　close　to　the　T1　copper　site　in　a　characterized　recombinant　laccase　from　Cerrena　unicolor　(rCuL).　Kinetic　data　for　computerassisted　virtual　mutants　established　the　binding　affinity　of　hydrogen　bonds　and　residues　(Asn336,　Asp207,　Val391,　and　Thr165)　in　rCuL　to　AFB1.　The　augmented　binding　affinity　to　AFB1　may　be　related　to　the　conformational　rearrangements　of　the　laccase　and　its　ability　to　hydrogen-bond　with　the　substrate.　Furthermore,　the　optimal　pH　and　temperature　for　rCuL　and　variants　mediated　AFB1　degradation　may　depend　on　their　pH　stability　and　thermostability.　Our　findings　reinforce　the　importance　of　the　structure-function　relationship　of　fungal　laccases　in　degrading　AFB1,　providing　mechanistic　guidance　for　future　biocatalyst　and　bioengineering　applications.