This paper reports the development of a novel monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein of the cephalochordate *Branchiostoma lanceolatum*. The protein exhibits exceptionally high quantum yield and extinction coefficient, making it a promising candidate for advanced imaging applications. Through directed evolution guided by structural modeling, 21 mutations were introduced into the wild-type protein to monomerize it. mNeonGreen shows sharp excitation and emission peaks, is brighter than most commonly used green and yellow fluorescent proteins, and has superior photostability. It performs well as a fusion tag for traditional imaging and stochastic single-molecule super-resolution imaging, and is an excellent Förster resonance energy transfer (FRET) acceptor for cyan fluorescent proteins. The study also highlights the potential of mNeonGreen for antibody development and orthogonal co-IP experiments.This paper reports the development of a novel monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein of the cephalochordate *Branchiostoma lanceolatum*. The protein exhibits exceptionally high quantum yield and extinction coefficient, making it a promising candidate for advanced imaging applications. Through directed evolution guided by structural modeling, 21 mutations were introduced into the wild-type protein to monomerize it. mNeonGreen shows sharp excitation and emission peaks, is brighter than most commonly used green and yellow fluorescent proteins, and has superior photostability. It performs well as a fusion tag for traditional imaging and stochastic single-molecule super-resolution imaging, and is an excellent Förster resonance energy transfer (FRET) acceptor for cyan fluorescent proteins. The study also highlights the potential of mNeonGreen for antibody development and orthogonal co-IP experiments.