Lactate is a key metabolite with applications ranging from monitoring training efficiency to early sepsis detection and monitoring the metabolic state of cell cultures. In this study, a paper-based lactate sensor utilizing a fluorescent readout was developed. Unlike common lactate dehydrogenase (LDH)-based methods, these sensors use a green fluorescent protein (GFP) or mApple-coupled lactate binding domain, which provides a fluorescent readout upon lactate binding. We demonstrate that immobilizing these proteins on paper does not affect their ability to bind lactate and produce a fluorescent readout, by monitoring lactate levels in the cell culture supernatant applying different cell culture conditions.