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Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3M and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger ste