Micro- and nanoporous systems incorporating bioactive molecules, such as enzymes, are very promising supports for biocatalysis. Here, we investigate the influence of geometrical confinement on the layer-bylayer (LbL) assembly of enzyme-based thin films, using the polyionic couple (chitosan/13-lactamase),. Thin films with different number of layers were prepared on flat silicon wafers and within cylindrical submicron pores of polycarbonate membranes to determine the impact of the confinement of macromolecules on: (i) the LbL film growth, (ii) the enzyme loading, and (iii) the biocatalytic efficiency. Solid-state NMR is employed to estimate the amount of enzyme loaded in the different types of LbL films, and the enzyme activity is determined by the study of the kinetics of nitrocefin hydrolysis. Film growth and loading of enzyme occur faster in the confined medium, until pores reach saturation. Moreover, when LbL films are grown within nanopores, the weight fraction of enzyme is very high and remains constant along the build-up. Conversely, the relative amount of enzyme in flat films significantly decreases with the number of layers due to the partial exchange during the growth. Finally, our study emphasizes that the immobilization of enzymes through LbL assembly in confined media can lead to very active surfaces with a restricted number of LbL cycles. (C) 2015 Elsevier B.V. All rights reserved.