Fluorescent proteins are essential and widely used molecular tools in biological research. T-Sapphire is a commonly employed green fluorescent protein with a large stokes shift (LSS), significantly reducing the absorption peak near 475 nm of the original GFP and retaining a peak at 389 nm. However, T-Sapphire still exhibits residual absorption between 475 nm and 500 nm, and its stability under acidic conditions requires improvement. We designed a fluorescent protein molecule—mJade based on superfolder GFP (sfGFP) gene sequence. Recombinant mJade was expressed, purified, and crystallized, and its crystal structure was resolved. We discovered that a unique hydrogen bond network near the chromophore of mJade leads to its large Stokes shift. By comparing the excitation spectra of mJade and T-Sapphire, we found that mJade significantly reduces absorption of light in the 475 500 nm range. Fluorescence intensity measurements of mJade under different pH conditions revealed excellent stability under alkaline conditions and significantly higher stability under acidic conditions compared to sfGFP and T-Sapphire. Bio-Layer Interferometry (BLI) experiments demonstrated that mJade maintains a high affinity for binding with the green fluorescent protein nanobody GBP1, which indicate that mJade can be used for simultaneous labeling of different molecules with EGFP and other fluorescent proteins to achieve multicolor tracking imaging.