Two new polyfluorenes with dipicolylamine (DPA) pendant, PF-TDPA and PF-HDPA, are designed and synthesized by pre-and post-functionalization, respectively. PF-TDPA with a rigid side chain shows a selective fluorescence quenching upon the addition of Cu2+ in a mixture solution of tetrahydrofuran and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. What is more, the PF-TDPA/Cu2+ complex can selectively detect histidine over other amino acids with a fluorescence recovery. In contrast, PF-HDPA with a flexible spacer exhibits a fluorescence quenching to Cu2+ but slightly fluorescence recovery after the addition of histidine. This indicates that the proper distance between the two DPA groups play an important role in the detection of histidine.