A series of hyper-branched cationic conjugated polyelectrolytes containing different contents of phosphorescent Ir(III) complex has been designed and synthesized successfully. Their photophysical properties in both aqueous solution and solid film are investigated and their morphologies in aqueous solution are observed by TEM. Nanoparticles with the size of 80-100 nm have been formed in aqueous solution through the self-assembly of polymers. An energy transfer process from host polyfluorene to guest Ir(III) complex exists and becomes more efficient in the solid films. Importantly, this series of hyper-branched polymers can be applied as light-up heparin probes with good selectivity, high sensitivity, and naked-eye detection through electrostatic interaction between polymers and heparin. Quantification for heparin in aqueous solution can be realized in the range of 0-44 μM in the buffer solution. Detection limit can reach as low as 50 nM. More meaningfully, time-resolved photoluminescence technique is utilized successfully in the heparin sensing to eliminate the background fluorescence interference effectively and enhance the sensing sensitivity in the complicated media.