Biosensors have gained significant attention in recent years since they can provide real-time, non-invasive, and highly sensitive detection of various biological targets. The materials used as platforms in optical biosensors play a crucial role in determining their performance, such as sensitivity, selectivity, stability, and response time. Metal-Organic Frameworks (MOFs) with Open Metal Sites (OMS) present a powerful platform for developing optical biosensors with increased functionalities. The combination of MOFs structural flexibility, large surface area, and the unique reactivity of OMS allows for highly sensitive, selective, and multiplexed detection of various biological targets, achieving the incorporation of biomolecules of different nature, to enable an increase in selectivity and specificity. These advantages make them highly suitable for applications in medical diagnostics, mainly focused on detecting glucose, pathogens, cancer biomarkers, toxins, and pollutant compounds, as we present in this revision. As research advances, we expect more innovative uses of functionalized MOFs-OMS in the optical biosensing field. Combining additional materials, such as biomolecules, nanoparticles, nanofibers, or quantum dots, enhances their output signal performance in biosensing applications for analyte detection in complex samples (i.e., clinical, serum, and food matrices). A direct comparison of the most critical properties of biosensors with and without MOFs-OMS is presented, where the detection of specific analytes often relies on changes in optical properties like fluorescence, absorption, or scattering. This review highlights the exceptional properties of MOFs with OMS, such as strong interaction with biomolecules, high sensitivity, and selectivity related to their nanostructural composition, describing the opportunities for future implementation in the diagnostics sector. Additionally, an outlook of forthcoming challenges underscores the potential of these materials for advancing novel optical biosensing technologies.