The invention provides a tissue optical clearing agent which comprises, in weight percent, 15-25% of urea, 8-15% of formamide, 10-20% of triton, 20-25% of N,N,N',N'-Four (2-hydroxy propyl) ethylenediamine and the rest water. According to the tissue optical clearing agent, biological tissue scattering can be effectively reduced in a short time, tissue transparency is achieved, the integrity of biological tissues can be effectively kept, the volume of the biological tissues is basically unchanged, expansion or contraction is avoided, tissues cannot become brittle, so that imaging depth and signal-to-noise ratio are greatly improved, and endogenous fluorescent signal strength is basically unaffected. The invention further provides a preparation method and an application of the tissue optical clearing agent.