Structurally defined block conjugated polymers (SDBCPs) are characterized by unique photophysical and self-assembly properties. However, the potential of SDBCP for sensing application has seldomly been exploited. Herein, we present the synthesis and application of a novel type of SDBCP, P3EPT17-b-P3ImHT33, which is comprised of regio-regular diblock polythiophene and imidazole side chains. P3EPT17-b-P3ImHT33 can self-assemble into nanofibers in DMSO via crystallization-driven self-assembly, while it self-assembles into quasi-spherical micelles in water via hydrophilic/hydrophobic phase separation. By exploiting the strong coordination interaction between Cu2+ and imidazole group, P3EPT17-b-P3ImHT33-based nanoparticles (PT–CPNs) were used as an effective fluorescent nanoprobe for the selective and sensitive detection of Cu2+. The detection limit can be down to 0.13 nM. Furthermore, fluorescence imaging studies demonstrated that PT–CPNs can efficiently detect Cu2+ in living HeLa cells. © 2020 Elsevier B.V.