The immobilization of proteins on ultrananocrystalline diamond (UNCD) surfaces is a pivotal step in the development of diamond-based biosensors. We discuss protein immobilization strategies, focusing on their effects on protein functionality and stability. We highlight the advantages and limitations of various surface functionalization techniques, including conventional linker chemistry and photochemical methods, with an emphasis on the suitability of structured and modified UNCD surfaces. We present key experimental protocols that detail the preparation and functionalization of UNCD surfaces, and the subsequent immobilization of proteins such as green fluorescent protein (GFP), antibodies, nanobodies, and designed ankyrin repeat proteins (DARPins). The results demonstrate the efficacy of nanobodies for GFP capture, outperforming other antigen-binding proteins in terms of cost-effectiveness and binding efficiency. This chapter also evaluates the influence of surface structuring and blocking proteins on immobilization performance, underscoring the interplay between physical adsorption and covalent binding. Finally, the potential of diamond-based sensors is contextualized within broader applications, including their robustness and selectivity in biosensing. Our findings contribute to the optimization of diamond-based biosensor platforms, paving the way for more reliable, cost-effective, and sensitive tools.