Poly{[2,5-bis(3-sulfonatobutoxy)-1,4-phenylene sodium salt]-alt-(1,4-phenylene)}, which is an anionically charged, water-soluble poly(para-phenylene) derivative with aldehyde groups at both chain ends, is prepared via the Suzuki coupling reaction in order to develop a FRET energy donor, while simultaneously dual-fluorescence-patterning the protein. Regardless of the end-capping, the synthesized polymer exhibits a good solubility in water with an absorption maximum at 338 nm and a photoluminescence maximum at 417 nm, similar to those of the the end-capped polymer. The emission spectrum of the polymer overlaps the absorption spectrum of fluorescein, and therefore, the polymer can be used as an energy donor with fluorescein as the energy acceptor in the FRET mechanism. This polymer design not only takes advantage of the introduction of biotin at both chain ends (through a reaction with the aldehyde end groups) to realize the facile interaction with streptavidin, but also brings into play the electrostatic features of the anionic sulfonate groups to fabricate an electrostatic self-assembly with polycation for the pattern substrate. The micropattern of fluorescein-labeled streptavidin is fabricated on the polymer-coated substrate through micro-contact printing using a polydimethylsiloxane mold. As a result, the polymer substrate exhibits a dual fluorescence micropattern, which results from the blue emission color from the energy donor and the FRET-amplified green emission from the energy acceptor. The high-resolution patterning is carried out for the application of multiplexing by simultaneously imaging the patterned green-emitting fluorescein by FRET and the surrounding blue-emitting polymer according to an optical detection scheme.