<p>DNA-templated silver nanoclusters (D-AgNCs) have exhibited unique performance advantages for developing label-free fluorescence biosensors; however, most of them are constructed on various single-signal output strategies and how to enhance their detection sensitivity still faces a significant challenge. Herein, a novel crosslinked DNA network nanostructure decorated by chameleon D-AgNCs was developed for constructing an ultrasensitive label-free ratiometric fluorescence biosensing method. The DNA nanostructure was assembled from an aptamer recognition-triggered rolling circle amplification (RCA) reaction under the assistance of a quadruple-way DNA scaffold. This aptamer recognition also triggered a DNA walking reaction to highly release a D-AgNC with yellow fluorescence emission and a nonfluorescent D-AgNC. Through the proximity assembly of the two D-AgNCs at the DNA nanostructure to form a new D-AgNC with red fluorescence emission, the ratiometric signal transduction strategy was constructed for the method. Due to the RCA-assisted amplified assembly of the DNA nanostructure to capture abundant D-AgNCs and dramatically enhance the red fluorescence signal through its nanoconfinement effect, this method exhibited a very low detection limit of 6.55 fg mL⁻¹ for selectively assaying the model antibiotic of kanamycin in a six-order of magnitude wide linear range. In addition, the one-step homogeneous assay with no need to form a DNA hydrogel well simplified the assay manipulation and also effectively decreased the assay cost for practical applications.</p>