Nucleases play a crucial role in DNA replication, recombination and repair which are associated with cancers. Herein, we develop a four-color fluorescent probe for ratiometric detection of multiple nucleases. This four-color fluorescent probe consists of four fluorescent dyes connected by a DNA tetrahedral nanostructure with the involvement of multistep fluorescence resonance energy transfer (FRET). Based on the principle of self-assembly, the four-color fluorescent probe is constructed by integrating one acceptor with three spatially and spectrally distinct acceptors. A DNA tetrahedral nanostructure functions as a scaffold to link the acceptor dyes (i.e., diethylaminocoumarin (DEA), carboxyfluorescein (FAM), Texas Red, and Cy5). The fluorescence emissions of DEA, FAM, Texas Red and Cy5 can be observed through efficient multi-step energy transfer. This four-color fluorescent probe enables single excitation/four emissions, and it can be used for ratiometric detection of nucleases (i.e., XhoI, HindIII and KpnI) and the screening of nuclease inhibitors. Importantly, this four-color fluorescent probe can be further applied to discriminate multiple biomolecule targets by simply integrating the recognition sites of various biomolecules into the DNA tetrahedral nanostructure.