Using alternating layers of charged polymers, we have constructed reactive thin films by incorporating enzymes, specifically alkaline phosphatase (AP) and glucose oxidase (GOD), into multilayers of a polycation, branched polyethylenimine (PEI), and a polyanion, poly(styrenesulfonate) (PSS), supported on a glass substrate. Experiments using a quartz crystal microbalance (QCM) demonstrated that the films grew sequentially on a solid support, while X-ray photoelectron spectroscopy (XPS) further confirmed the growth and deposition of successive enzyme layers. For both enzymes, the reactive films demonstrated increased activity with the successive number of deposited enzyme layers. In hybrid films, consisting of alternating layers of AP and GOD, both enzymes retained activities similar to those of their corresponding films of either enzyme alone. The effect of elevated temperature was also investigated for these reactive films. Increased thermal stability was found to be associated with the increase in the number of deposited enzyme layers.