Color pallet of fluorescent protein (FP) has made a great contribution to visualize molecular and cellular processes. However, most FPs lose fluorescence at pH lower than their neutral pKa (≈ 6), and this has hampered their application in acidic organelles such as endosomes, secretory granules, lysosomes and vacuoles (pH ≈ 4.5-6.0). To date, several acid-tolerant FPs are available for cyan and red color, however there is little report of acid-tolerant green FPs (GFPs) practically applicable to bioimaging. Here we developed an acid-tolerant monomeric GFP “Gamillus” from jellyfish Olindias formosa with excellent brightness, maturation speed and photostability. Results of X-ray crystallography and point mutagenesis suggest that the acid-tolerance is attributed to stabilization of deprotonation on chromophore phenyl ring in broad pH range by forming unique trans configuration. We demonstrated that Gamillus serves as a universal molecular tag, suitable for imaging in acidic organelles through autophagy-mediated molecular tracking to lysosomes. Multicolor imaging in combination of Gamillus with reported color pallet of acid-tolerant FPs is expected to unveil physiological phenomena in acidic environments.