The applications of conjugated polyelectrolytes in biosensing and bioimaging have attracted more and more research interests due to their excellent photophysical properties. In this work, a new series of conjugated polyelectrolytes containing long-lived phosphorescent Ir(III) complexes is designed and synthesized, which can be used for ratiometric and lifetime-based sensing of heparin utilizing the electrostatic interaction between cationic polymers and anionic heparin. By changing the ligand structures of Ir(III) complexes, the sensing performances of phosphorescent-conjugated polyelectrolytes (PCPEs) are optimized. In addition, the application of PCPEs in cellular imaging is carried out. These polymers can be applied for specific staining of cell membrane. Importantly, utilizing the long emission lifetime of phosphorescent signal of Ir(III) complexes, time-gated luminescent imaging is carried out, which can eliminate the short-lived background fluorescence interferences from the environment or biological samples.