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The detection of biomarkers is of great significance in the diagnosis of numerous diseases, especially cancer. Herein, we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads, DNA G-quadruplex, and exonuclease Ⅲ (Exo Ⅲ). In the presence of a target protein, a label-free single strand DNA (ssDNA) hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target. Subsequently, ssDNA initiates the Exo Ⅲ-aided recycling to amplify the fluorescence signal, which was caused by N-methylmesoporphyrin IX (NMM) insertion into the G-quadruplex structure. This proposed strategy combines the excellent specificity between the aptamer and target, high sensitivity of the fluorescence signal by G-quadruplex and Exo Ⅲ-aided recycling amplification. We selected (50–1200 nmol/L) MUC1, a common tumor biomarker, as the proof-of-concept target to test the specificity of our aptasensor. Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection (LODs) of 3.68 and 12.83 nmol/L in buffer solution and 10％serum system, respectively. The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.