We present a novel ratiometric fluorescent biosensor for ctDNA analysis based on the construction of a DNA four-way junction (FWJ). Three fuel strands for the FWJ are firstly designed and prepared. Another essential strand for the formation of the structure is the DNA product generated from target ctDNA initiated strand displacement amplification. With the transformation of the DNA structure, the FRET states of two fluorophores change and the ratiometric fluorescence response can be recorded to indicate the level of the initial ctDNA. The proposed method also has excellent capability to discriminate mismatches and shows potential practical utility for clinical samples.