Zinc oxide (ZnO) is a wide bandgap semiconductor material that has been widely explored for countless applications, including in biosensing. Among its interesting properties, its remarkable photoluminescence (PL), which typically exhibits an intense signal at room temperature (RT), arises as an extremely appealing alternative transduction approach due to the high sensitivity of its surface properties, providing high sensitivity and selectivity to the sensors relying on luminescence output. Therefore, even though not widely explored, in recent years some studies have been devoted to the use of the PL features of ZnO as an optical transducer for detection and quantification of specific analytes. Hence, in the present paper, we revised the works that have been published in the last few years concerning the use of ZnO nanostructures as the transducer element in different types of PL-based biosensors, namely enzymatic and immunosensors, towards the detection of analytes relevant for health and environment, like antibiotics, glucose, bacteria, virus or even tumor biomarkers. A comprehensive discussion on the possible physical mechanisms that rule the optical sensing response is also provided, as well as a warning regarding the effect that the buffer solution may play on the sensing experiments, as it was seen that the use of phosphate-containing solutions significantly affects the stability of the ZnO nanostructures, which may conduct to misleading interpretations of the sensing results and unreliable conclusions.