MXene Ti3C2, as a new class of two-dimensional (2D) transition metal carbides (or nitrides), has been synthesized by exfoliating pristine Ti3AlC2 phases with hydrofluoric acid. The SEM and XRD images show that the resultant MXene possesses a graphene-like 2D nanostructure, and the surface of MXene has been partially terminated with -OH, thus providing a favorable microenvironment for enzyme immobilization and retaining their bioactivity and stability. Considering the unique metallic conductivity, biocompatibility and good dispersion in aqueous phase, the as-prepared MXene was explored as a new matrix to immobilize tyrosinase (a model enzyme) for fabricating a mediator-free biosensor for ultrasensitive and rapid detection of phenol. The varying electrochemical measurements were used to investigate the electrochemical performance of MXene-based tyrosinase biosensors. The results revealed that the direct electron transfer between tyrosinase and electrode could be easily achieved via a surface-controlled electrochemical process. The fabricated MXene-based tyrosinase biosensors exhibited good analytical performance over a wide linear range from 0.05 to 15.5 μmol L-1, with a low detection limit of 12 nmol L-1 and a sensitivity of 414.4 mA M-1. The proposed biosensing approach also demonstrated good repeatability, reproducibility, long-term stability and high recovery for phenol detection in real water samples. With those excellent performances, MXene with graphene-like structure is proved to be a robust and versatile electrochemical biosensing platform for enzyme-based biosensors and biocatalysis, and has wide potential applications in biomedical detection and environmental analysis.