A double fluorescence mu-well assay that exploits a novel conjugated polymer containing cyclodextrin (CD) as the key component is reported. For the construction of the cocaine bioassay, poly(p-phenylene) with CD units in the main-chain and poly(ethylene glycol) side chains (PPP-CD-g-PEG) was first prepared by Suzuki coupling polymerization and coated on each well as a biomimetic material. Although the poly-phenylene backbone is responsible for the fluorescence properties without an additional fluorophore, PEG and CD provide water solubility and selective complexation with cocaine, respectively. A cocaine antibody was used as a secondary recognition compound after labelling with quantum dots (QDs). Most notably, we show that the two-color fluorescence nature of the assay facilitates double measurement from the same sample and the described strategy can be adapted to various sensing systems.