A　＂turn-on＂　and　label-free　fluorescent　assay　for　the　specific,　rapid,　and　sensitive　detection　of　3＇aEuro　parts　per　thousand　a　dagger＇aEuro　parts　per　thousand　5＇　exonuclease　III　activity　is　reported　in　this　study.　The　assay　is　based　on　the　Tb3+-promoted　G-quadruplex,　which　lead　to　the　enhancement　of　Tb3+　fluorescence　due　to　the　energy　transfer　from　guanines.　The　proposed　assay　is　highly　simple,　rapid,　and　cost-effective,　and　does　not　require　sophisticated　experimental　techniques　such　as　gel-based　equipment　or　radioactive　labels.　It　can　be　used　for　the　rapid　detection　of　exonuclease　III　activity　with　a　detection　limit　of　0.8　U　and　a　RSD　(n　=　6)　＜　5　%.　Notably,　no　dye　was　covalently　conjugated　to　the　DNA　strands,　which　offers　the　advantages　of　low-cost　and　being　interference-free.