Oriented enzyme immobilisation has become a crucial parameter in the rational design of biosensors for clinical analysis, food technology and environmental applications. The possibility to control the biomolecule orientation eliminates the enzyme deactivation and/or active site blocking, opening the door to more sensitive, reliable and stable devices. Nowadays, different techniques are available to created highly ordered structures on biosensing supports, each one of them presenting their own advantages and drawbacks. Some of these immobilisation techniques are based on affinity interactions between biomolecules, such as enzymes and antibodies, biotin and (strept)avidin molecules, and lectins and sugars. Other strategies take advantage of the carbohydrate chains present on the enzyme surface and their reactions with hydrazides or boronic acids. The thiol – thiol or thiol – gold binding and the interaction between certain aminoacid residues, e.g. Cys and His, and divalent metal ions have been also exploited to achieve ordered enzyme configurations. This article summarises these strategies and reports our contribution to the field.