The invention provides a fluorescent protein pair for highly sensitive FRET imaging and application thereof. Specifically, a red fluorescent protein is provided. Compared with the amino acid sequenceof mRuby2, the amino acid sequence of the red fluorescent protein has the following mutation sites: N33R, M36E, T38V, K74A, G75D, M105T, C114E, H118N, Q120K, H159D, M160I, S171H, S173N, I192V, L202I,M209T, F210Y, H216V, F221Y, A222S and G223N. The invention also provides a green fluorescent protein with the amino acid sequence having the following mutation sites compared to the amino acid sequence of Clover: N149Y, G160S or G160C and A206K. The red fluorescent protein and the green fluorescent protein have good photostability and can be used for highly sensitive FRET imaging.