In this work, homo-FRET (Förster resonance energy transfer between the same kind of fluorophores) takes place in a hetero-FRET (FRET between two different fluorophores) system and can effectively improve the energy transfer efficiency. Herein, a novel ratiometric fluorescence method was developed for the detection of nuclease activity. Exonuclease III (Exo III), an enzyme which has a high exodeoxyribonuclease activity for double-stranded DNA (dsDNA) in the 3' to 5' direction, was chosen as a proof of concept of this strategy. In a linear dsDNA template, the occurrence of homo-FRET in two Cy3 donors enables the highly efficient transfer of energy to the Cy5 acceptor. The ratio of fluorescence intensity between Cy3 and Cy5 (FD/FA) increases in an Exo III concentration-dependent manner, which built the foundation of Exo III quantification. This method exhibits a linear range from 0.25 to 8 U mL-1 with a detection limit of 0.17 U mL-1. Importantly, this platform also shows the potential for screening Exo III inhibitors and detecting Exo III activity in complex samples.