A　large　amount　of　antibiotics　enters　the　soil　environment　and　accumulates　therein　as　individuals　and　mixtures,　threatening　the　soil　safety.　However,　there　is　little　information　regarding　the　influence　of　single　and　mixed　antibiotics　on　key　soil　proteins　at　molecular　level.　In　this　study,　setting　sulfadiazine　(SD)　and　tetracycline　hydrochloride　(TC)　as　the　representative　antibiotics,　the　interactions　between　these　agents　and　α-amylase　(an　important　hydrolase　in　soil　carbon　cycle)　were　investigated　through　multi-spectroscopic　approaches,　X-ray　photoelectron　spectrometry,　and　molecular　modeling.　It　was　found　that　both　SD　and　TC　spontaneously　bound　to　α-amylase　with　1:1　stoichiometry　mainly　via　forming　stable　chemical　bonds.　The　interactions　altered　the　polarity　of　aromatic　amino　acids,　protein　backbone,　secondary　structure,　hydrophobicity　and　activity　of　α-amylase.　The　SD-TC　mixtures　were　designed　based　on　the　direct　equipartition　ray　to　comprehensively　characterize　the　possible　concentration　distribution,　and　interactive　effects　indicated　that　the　mixtures　antagonistically　impacted　α-amylase.　These　findings　reveal　the　binding　characteristics　between　α-amylase　and　typical　antibiotics,　which　probably　influence　the　ecological　functions　of　α-amylase　in　soil.　This　study　clarifies　the　potential　harm　of　antibiotics　on　soil　functional　enzyme,　which　is　significant　for　the　environmental　risk　assessment　of　antibiotics　and　their　mixtures.　©　2024　Elsevier　B.V.