A new fluorescent microgel containing the CP moiety (PFP-NIPAm) was prepared with the size within 100 nm. Covalent linking of the conjugated polyelectrolyte moiety into the microgel prevents leakage of the fluorophore while keeping its fluorescence property. The new fluorescent microgel can be used as an optical platform to detect DNA and enzyme. More importantly, because the electrostatic attraction dominates the interactions between cationic PFP-NIPAm microgel and negatively charged target, washing with high ionic strength aqueous solution can block their interactions and displace the target from the PFP-NIPAm microgel. Thus, PFP-NIPAm can be readily reusable for detection. Another unique feature is that the PFP-NIPAm microgel extends the detection media from homogeneous solution to solid phase, which shows great potential for biodetection in the real world using conjugated polymers. Furthermore, the detection can be carried out under UV light, and no expensive detection instrumentation is needed. In principle, such an assay system could be expanded to a high-throughput assay.