Fluorescent proteins (FPs) are luminescent biomolecules that emit characteristic hues upon irradiation. A group of calmodulin (CaM)-green FP (GFP) chimeras have been previously engineered to enable the optical detection of calcium ions (Ca2+). We investigate one of these genetically encoded Ca2+ biosensors for optical imaging (GECOs), GEM-GECO1, which fluoresces green without Ca2+ but blue with Ca2+, using femtosecond stimulated Raman spectroscopy (FSRS). The time-resolved FSRS data (<800 cm1) reveal that initial structural evolution following 400 nm photoexcitation involves small-scale coherent proton motions on both ends of the chromophore two-ring system with a <250 fs time constant. Upon Ca2+ binding, the chromophore adopts a more twisted conformation in the protein pocket with increased hydrophobicity, which inhibits excited-state proton transfer (ESPT) by effectively trapping the protonated chromophore in S1. Both the chromophore photoacidity and local environment form the ultrafast structural dynamics basis for the dual-emission properties of GEM-GECO1. Its photochemical transformations along multidimensional reaction coordinates are evinced by distinct stages of FSRS spectral evolution, particularly related to the similar to 460 and 504 cm(1) modes. The direct observation of lower frequency modes provides crucial information about the nuclear motions preceding ESPT, which enriches our understanding of photochemistry and enables the rational design of new biosensors.