In this paper, a novel ratiometric and cascade amplification strategy was developed by combining the unique signal amplification and effective fluorescence resonance energy transfer (FRET) property of conjugated polymers with the Exo III-assisted target recycling method. The target DNA (ssDNAc) could be hybridized with the duplex-stranded probe to trigger the cyclic digestion of the probe strands and lead to the continuous release of fluorescein from the probe. The proposed strategy thus shows enhanced sensitivity toward target DNA with a detection limit of 0.38 nM, which is more sensitive than the previously reported comparable biosensors based on conjugated polyelectrolytes. Furthermore, this method exhibited an improved performance to discriminate single mismatched targets through an efficient FRET-based ratiometric detection method using a conjugated polymer as a donor and an optical transducer. More importantly, this cascade amplification approach offers the advantages of simplicity, which avoids multiple utilization of probes and complex assay steps required in traditional amplification methods.