A drug delivery system that is integrated with fluorescent imaging is an emerging platform for tumor diagnostic and therapy. A pH-responsive fluorescent polymer that can respond to the surrounding medium is a desired component with which to construct an advanced drug delivery system with bioimaging characteristics and controllable drug releasing. In this work, we synthesized novel amphiphilic block copolymers of poly(ethylene glycol)-b-poly(2-(diisopropylamino) ethyl methacrylate-co-dithiomaleimide) (PEG-b-poly(DPA-co-DTM)) and poly(ethylene glycol)-b-poly(2-(dibutylamino) ethyl methacrylate-co-dithiomaleimide) (PEG-b-poly(DBA-co-DTM)) with pH-responsiveness and fluorescence. The block copolymers exhibited relatively stable fluorescence properties in different solvent and excitation-independent fluorescence behaviours. By copolymerizing the responsive segments in the molecule chain, the doxorubicin (DOX)-loaded micelles could be triggered to disassemble, thus releasing DOX at the corresponding pH values and yielding a pH-responsive drug release. Targeted deliveries of the drug within the cell were demonstrated by using the carrier responding to different pH values. The best antitumor effect was obtained by PEG-b-poly(DPA-co-DTM), which immediately released DOX as soon as it entered the tumor cells, as a result of responding to the regional pH level (pH = 6.3). The pH-responsive copolymers showed excellent biocompatibilities, as nearly 85% of cells with these fluorescent micelles survive when the testing concentration goes up to 200 μg mL−1. In all, these pH-responsive and dithiomaleimide-based fluorescent block copolymers hold great potential in future cancer diagnostic and therapeutic techniques.