Laccase-catalyzed oxidative reactions are increasingly examined as a reliable approach to environmental analysis and remediation, and it is urgent to widen metal category to compensate huge gap in the number of studies on copper- and non-copper laccase mimics. Herein, two-dimensional ultrathin MnO2 nanofilm (Mn-uNF) was designed via a chemical deposition and alkali etching process. Similar to Cu-laccase, Mn-uNF can oxidize phenols via a one-electron-transfer reaction of Mn(III) and accelerate the MnIII/MnIV state cycle through an unconventional oxygen reduction process. The excellent laccase-like performance of Mn-uNF can be ascribed to the abundant atomically dispersed Vo-assisted Mn(III) and surface -OH species, which was confirmed by characterizations and DFT calculation. Further, a facile dual-function colorimetric platform was designed for array sensing of o-, m-, and p-dihydroxybenzene isomers and one-step discrimination of tetracyclines containing phenol groups. These findings provide reasonable guidance for the design of a nanozyme with active Mn sites as a new family member of highly efficient copper-free laccase mimics. Display omitted •Ultrathin MnO2 designed as a new member of highly efficient copper-free laccase mimics.•A new synergy between Vo-assisted MnIII and -OH activated by an alkali etching process.•One-step discrimination of TC, OTC, CTC based on laccase mechanism for the first time.•A facile colorimetric sensor for selective recognition of o-, m-, and p-dihydroxybenzene isomers.