Tyrosinase (TYR) is a multifunctional copper-containing enzyme that plays a critical role in the biosynthetic pathway of melanin. Thus, the detection of TYR activity possesses vast importance from clinical diagnosis to the food industry. However, most TYR detection methods are expensive, complicated, and time-consuming. Herein, a functional nanofluidic heterochannel composed of an ultrathin tyramine-modified mesoporous silica layer (Tyr-MS) and alumina oxide (AAO) arrays is constructed by an interfacial super-assembly method. The heterochannel with plenty of enzyme catalytic sites for TYR provides the response of the ion current signal against TYR concentrations. Introducing enzymatic reaction paves the way for the heterochannel to achieve label-free, selective, specific detection of TYR. Notably, a highly sensitive detection of TYR with a limit of 2 U mL-1 was obtained by optimizing the modified conditions. Detailed investigations and theoretical calculations further reveal the mechanism for the detection performance. This work provides a simple, low-cost, quick response, and label-free platform based on functional nanofluidic devices for enzyme-sensing technologies.