A novel monomeric yellow-green fluorescent protein, mNeonGreen, was derived from a tetrameric fluorescent protein in the cephalochordate Branchiostoma lanceolatum. This protein is the brightest monomeric green or yellow fluorescent protein described, with high quantum yield and extinction coefficient, making it significantly brighter than most commonly used GFPs and YFPs. It exhibits excellent photostability and is a good FRET acceptor for cyan fluorescent proteins. mNeonGreen performs well as a fusion tag for traditional and superresolution imaging and is suitable for FRET applications. It is also highly useful for general imaging, FRET probe design, and single-molecule superresolution imaging. The protein was engineered through directed evolution to monomerize the tetrameric LanYFP. mNeonGreen has superior optical properties compared to other fluorescent proteins and is expected to be highly useful for various applications. It is also an attractive target for antibody development and can be used in orthogonal co-IP experiments with other fluorescent proteins. The study also reports the performance of mNeonGreen in live cell imaging and superresolution imaging, showing its effectiveness in various cellular structures and applications. The protein was characterized using various techniques, including spectroscopy, size exclusion chromatography, and fluorescence lifetime imaging. The study also compares mNeonGreen with other fluorescent proteins, such as Clover, and highlights its advantages in terms of photostability, maturation time, and monomeric character. The results demonstrate that mNeonGreen is a promising fluorescent protein for a wide range of applications in biological imaging and research.A novel monomeric yellow-green fluorescent protein, mNeonGreen, was derived from a tetrameric fluorescent protein in the cephalochordate Branchiostoma lanceolatum. This protein is the brightest monomeric green or yellow fluorescent protein described, with high quantum yield and extinction coefficient, making it significantly brighter than most commonly used GFPs and YFPs. It exhibits excellent photostability and is a good FRET acceptor for cyan fluorescent proteins. mNeonGreen performs well as a fusion tag for traditional and superresolution imaging and is suitable for FRET applications. It is also highly useful for general imaging, FRET probe design, and single-molecule superresolution imaging. The protein was engineered through directed evolution to monomerize the tetrameric LanYFP. mNeonGreen has superior optical properties compared to other fluorescent proteins and is expected to be highly useful for various applications. It is also an attractive target for antibody development and can be used in orthogonal co-IP experiments with other fluorescent proteins. The study also reports the performance of mNeonGreen in live cell imaging and superresolution imaging, showing its effectiveness in various cellular structures and applications. The protein was characterized using various techniques, including spectroscopy, size exclusion chromatography, and fluorescence lifetime imaging. The study also compares mNeonGreen with other fluorescent proteins, such as Clover, and highlights its advantages in terms of photostability, maturation time, and monomeric character. The results demonstrate that mNeonGreen is a promising fluorescent protein for a wide range of applications in biological imaging and research.