The scope of this thesis is the synthesis of nanostructured materials, their functionalization and use for optical lactate biosensing applications. Rapid detection of L-lactate is important in many applications in the clinical sector, in the food industry, or in biotechnology. The formation of enzyme loaded nanostructured materials is a promising approach to obtain performing, reliable and stable enzyme-based optical biosensors. Two different sensing schemes are proposed: the development of lactate-responsive films (pathches) and the development of a microparticle based lactate detection system. The content of this work can be divided in three main tasks: (i) the synthesis of nanostructured support materials for enzyme immobilization, (ii) the functionalization of these materials towards lactate detection, and (iii) the assessment of the performance and sensitivity of these enzyme-loaded films and particles for biosensor applications. The design of porous supports aims at providing large surface area for enzyme loading. Additionally, the porosity design must take into account that enzymes are large biomolecules, requiring tailored pore sizes to be transported and immobilized within the inner surface of the material. In this work, the synthesis of large mesoporous silica films with pore sizes ranging from 10 to 50 nm was explored by two different methodologies: i) a bottom up approach was followed using amphiphilic block copolymers as porogenic templates and silicon alkoxides as silica precursors, and ii) a powder approach using pre-synthesized silica particles in form of agglomerates was employed to obtain stable silica dispersions, which after casting resulted in porous silica films with pore sizes within the large-mesopore range. Multiscale porous films including large mesopores and macropores were synthesised by the powder approach, where polymer particles as additional macroporous templates were used. The functionalization of nanostructured silica films for L-lactate biosensing was explored using two different enzymes. As a first alternative, lactate dehydrogenase (LDH) was immobilized on amino-functionalized silica surfaces, and its immobilization and performance towards lactate detection was compared between mesoporous and multiscale porous films. Multiscale porous enzyme supports increased enzyme loading, offering better stability, and stronger and faster response compared to mesoporous-only films, allowing more sensitive and more robust detection of L-lactate.A novel concept for L-lactate biosensing is finally presented using a second enzyme, lactate oxidase (LOx); unlike LDH, it does not require a cofactor. This detection scheme is based on a combination of a powder approach for the synthesis of the enzyme silica support and a layer-by-layer assembly of polyelectrolytes. These hybrid systems incorporate cytochrome C (CytC) as a biosensing element. Its optical properties depend on its oxidation state andcan be coupled to the oxidation of L-lactate via the generation of hydrogen peroxide by LOx. In addition to the film-based detection schemes, a final part of the thesis focuses on the formation of LOx-CytC particles using porous calcium carbonate particles as sacrificial templates. The resulting protein-based particles can be used directly for facile optical detection of lactate; moreover, they are interesting materials for future developments of optical detection methods based on liquid suspensions, for example in cell media.