In vivo, enzymatic reactions occur in confined environments. Such conditions can strongly improve enzyme behavior and are therefore interesting to study for further applications in biocatalysis. Here, we report on the influence of nanoconfinement on the catalytic properties of enzyme-based nanotubes, built by the layer-by-layer (LbL) assembly of branched polyethylenimine (bPEI) and glucose oxidase (GOx) in nanoporous polycarbonate membranes (PCm). More precisely, the influence of nanoconfinement on the biocatalytic activity is investigated by varying the number of (bPEI/GOx) bilayers, the concentration of polyelectrolytes (PEs) used for LbL deposition and the pore diameter of the PC membrane. Bicinchoninic acid (BCA) assay is employed to estimate the amount of enzyme loaded in the different LbL assemblies. The enzymatic activity was monitored, and found to depend on the three studied parameters. Typically, it decreases with decreasing pore diameter under high concentration of PEs, which may be attributed to limitations of substrate/product diffusion within the network formed in small pores. However, when lower concentration of PEs is used for the LbL assembly, the assemblies features a different macromolecular distribution and the enzymatic activity becomes optimal for low pore diameters. The results of this study pave the way to a more rational design of enzyme-loaded porous nanostructures for biocatalysis.