An antibiotic-imprinted cavity with two different fluorescent dyes was prepared by molecular imprinting and subsequent post-imprinting modifications (PIMs), for the readout of a specific binding event as a fluorescence signal. The fluorescent dyes were site-specifically introduced into the cavity using an orthogonal reversible bonding reaction, Schiff base formation, and a disulfide exchange reaction. The template molecule, comprising cephalexin connected to a Schiff base monomer and a disulfide monomer, was copolymerized with a crosslinker. This was followed by Schiff base hydrolysis and a disulfide exchange reaction, yielding the APO-type scaffold (PRECURSOR). Two different fluorophores, 4-formylsalicylic acid (FSA) and 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD), were introduced into the PRECURSOR by site-directed PIMs, yielding a fluorescent HOLO polymer. The obtained fluorescent polymer, HOLO(FSA)(Cys-DBD), was able to selectively readout the binding events as a fluorescence signal change. We found that the proposed strategy has the potential to open up a new class of synthetic materials possessing various desirable functions.