Since being discovered in 2011, a large class of two-dimensional materials, labeled MXenes, has received increased research enthusiasm both theoretically and experimentally due to the unique physical, optical and electrical properties. Here, we prepared few-layered Ti₃C₂ nanosheets by a facile two-step liquid exfoliation method and, for the first time, demonstrated their intrinsic peroxidase-like activity in a Ti₃C₂–TMB–H₂O₂ system. The as-produced Ti₃C₂ nanosheets, especially after histidine modification, were characterized with excellent water dispersibility, large specific surface area, and high stability, which contribute to their much higher affinity to both substrates when compared to HRP. We have also established the catalytic mechanism whereby Ti₃C₂ nanosheets, where Ti switched spontaneously from an oxidized to reduced state, promoted the electron transfer from TMB to H₂O₂. Given the color reaction of Ti₃C₂ nanosheets, we have fabricated a colorimetric paper-based sensor integrated with a smartphone to detect glucose and an immunoassay to detect IR-β, enabling Ti₃C₂ nanosheets to be a powerful tool in the biodetection field.