Green Fluorescent Protein (GFP) is a biomolecule with natural and intense fluorescence, which is very promising for biological and medical applications. Although GFP is widely used as a biomarker, the application of this protein as a biosensor is still limited by its high costs and lack of knowledge about its stability in the presence of different conditions and stress. While there are reports indicating the range of stability of GFP in pH, most studies only show the presence or lack of fluorescence of the protein, with no in-depth structural evaluations or analysis of the process’ reversibility, crucial for the application of this protein as a biosensor. In this study, we evaluated the activity of GFP by 3D fluorescence spectroscopy and its secondary structure by circular dichroism. GFP was exposed to different pH for 30 minutes and the pH was returned to neutrality (7.4), to evaluate if the loss of fluorescence was reversible. The behaviour was highly pH dependent. Above pH 7.4 there was fluorescence increase, but the secondary structure was maintained. At pH 6 the fluorescence intensity of GFP decreased by half, had a shift in the maximum fluorescence peak position, but had no observable change to its secondary structure. At pH 5, there was modification in the secondary structure of GFP, but it was mostly reversible after the return to pH 7.4. However, at pH 4 and below, GFP presented massive structural change, and both fluorescence and secondary structure were not recovered after the return to pH 7.4. This data can contribute for the design of GFP biosensors and help to elucidate the processes controlling protein structural stability. Acknowledgments: FAPESP (2014/19793-3, 2014/16424-7, 2018/50009-8, 2018/06576-5, 18/01858-2), CAPES, CNPq, RMIT University.