Acetaminophen　(APAP)　is　widely　used　for　its　antipyretic　properties　and　is　commonly　found　in　wastewater,　potentially　threatening　aquatic　ecosystems.　Microbial　degradation　has　been　proven　effective　in　removing　APAP.　However,　strains　capable　of　efficiently　degrading　APAP　are　rare,　and　the　research　on　the　biodegradation　kinetics　of　APAP　is　insufficient.　In　this　study,　a　novel　APAP-degrading　strain,　identified　as　Delftia　sp.　PY-12　by　morphological　characterization　and　16S　rRNA　gene　sequencing,　was　isolated　from　activated　sludge.　Experimental　conditions　were　optimized　using　the　Box-Behnken　response　surface　design,　revealing　that　PY-12　degraded　100　mg/L　APAP　under　pH　7.11,　30　degrees　C,　and　163　rpm　within　24　h,　outperforming　most　other　strains.　Comparative　analysis　of　substrate　inhibition　models　shows　that　the　Aiba　and　Tiesser　were　most　suitable　for　the　substrate　inhibition　characteristics　of　PY-12.　The　models　indicate　that　PY-12　can　still　degrade　APAP　even　at　concentrations　of　446.919　mg/L　and　554.215　mg/L.　Through　potential　enzyme　activity　measurements,　the　degradation　pathway　of　APAP　by　PY-12　was　determined.　APAP　is　first　converted　by　amidohydrolase　into　4-aminophenol　(4-AP)　and　then　converted　by　deaminase　into　hydroquinone　(HQ),　which　is　further　converted　by　hydroquinone　1,2-dioxygenase　to　form　4-hydroxymuconic　semialdehyde,　ultimately　entering　the　TCA　cycle.　This　study　revealed　the　potential　application　value　of　PY-12　in　APAP　degradation,　which　could　be　a　strong　candidate　strain　for　microbial　bioremediation　of　APAP　pollution　in　environmental　remediation.