Abstract Persistent luminescence nanoparticles (PLNPs) are innovative materials able to emit light for a long time after the end of their excitation. Thanks to this property, their detection can be separated in time from the excitation, making it possible to obtain images with a high signal‐to‐noise ratio. This optical property can be of particular interest for the development of in vitro biosensors. Here, we report the unexpected effect of hydrogen peroxide (H 2 O 2 ) on the signal intensity of ZnGa 2 O 4 :Cr 3+ (ZGO) nanoparticles. In the presence of H 2 O 2 , the signal intensity of ZGO can be amplified. This signal amplification can be used to detect and quantify H 2 O 2 in various media, using non‐functionalized ZGO nanoparticles. This small molecule can be produced by several oxidases when they react with their substrate. Indeed, the quantification of glucose, lactic acid, and uric acid is possible. The limit of detection could be lowered by modifying the nanoparticles synthesis route. These optimized nanoparticles can also be used as new biosensor to detect larger molecules such as antigen, using the appropriate antibody. This unique property, i.e., persistent luminescence signal enhancement induced by H 2 O 2 , represents a new way to detect biomolecules which could lead to a very large number of bioassay applications.