AbstractA supramolecular assembly scheme is developed to enable the facile in‐situ immobilization of enzymes in a microfluidic channel system. A combination of orthogonal supramolecular interactions of host (β‐cyclodextrin)–guest (adamantane) and biotin–Streptavidin (SAv) interactions are employed to generate reusable homogeneous enzyme layers in microchannels. The structural integrity and catalytic activity of the immobilized enzyme calf‐intestine alkaline phosphatase (AlkPh) is demonstrated. From the kinetic analysis of a dephosphorylation reaction, the specificity constant kcat/KM for immobilized alkaline phosphatase in the channels is on the order of 105 M−1s−1 and comparable to known literature values in other environments. These observations are ascribed to the good access of the substrate to favorably oriented enzymes across the microchannel. Therefore, this study demonstrates the great potential for adopting a supramolecular assembly scheme to immobilize enzymes in microfluidic devices.