The activity of certain proteolytic enzymes is often an indicator of disease states such as cancer(1,2), stroke(2) and neurodegeneracy(3,4), so there is a need for rapid assays that can characterize the kinetics and substrate specificity of enzymatic reactions. Nanostructured membranes can efficiently separate biomolecules(5), but coupling a sensitive detection method to such a membrane remains difficult. Here, we demonstrate a single mesoporous nanoreactor that can isolate and quantify in real time the reaction products of proteases. The reactor consists of two layers of porous films electrochemically prepared from crystalline silicon. The upper layer, with large pore sizes (similar to 100 nm in diameter), traps the protease and acts as the reactor. The lower layer, with smaller pore sizes (similar to 6 nm), excludes the proteases and other large proteins and captures the reaction products. Infiltration of the digested fragments into the lower layer produces a measurable change in optical reflectivity, and this allows label-free quantification of enzyme kinetics in real time within a volume of similar to 5 nm.