The exponential development of biosensors as powerful analytical tools in the last four decades mainly relies on the high sensitivity and selectivity offered when detecting the target analyte. The transducer and the biological receptor are the bases of the biosensor development. Nevertheless, the bioreceptor immobilisation is also important, playing a key role in the retention of the biological activity, and thus affecting the sensitivity. Parameters such as shelf-life and surface regeneration also depend on the biomolecule immobilisation. Researchers are focusing their efforts towards random and oriented immobilisation procedures. Adsorption, entrapment, cross-linking and electrostatic interactions provide randomly immobilised biomolecules, sometimes partially hindering their biological activity. Covalent binding and affinity interactions may enable oriented biomolecule immobilisations, providing controlled, reproducible and highly active modified surfaces. This paper reviews the main immobilisation strategies used in the biosensors development, putting special emphasis on our contribution to mild and oriented immobilisation techniques.