Inspired by nature, researchers aim at bringing together different types of enzymes by the generation of multi-enzymatic structures. Amongst others, chemical methods have been exploited enabling the covalent linkage of a set of enzymes to the same macromolecular scaffold or direct cross-linking. Control over the relative position of enzymes in the system has been realized by sequential immobilization in microchannels and by positional co-localization on DNA nanostructures. So far, site-specific conjugation reactions such as the azide-alkyne cycloaddition, N-terminal transamination and enzyme-mediated cross-linking, have been applied to a limited extent only. These methods are expected to allow for co-immobilization of less robust enzymes, hence, an expansion in the diversity of immobilized biocatalytic cascades. In addition, the combination of multiple bioconjugation methods will provide control over the composition in scaffold-free multi-enzyme complexes.