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We report for the first time the use of time-resolved technique in biodetection based on the phosphorescent conjugated polyelectrolytes (PCPEs) in both aqueous environment and cell to eliminate autofluorescence and enhance S/N ratio.A PCPE-based heparin probe with low detection limit,high selectivity and naked-eye detection is realized.Quantifications for heparin are achieved in the range of 0-70 μM and 0-5 μM in water and in serum.This PCPE-based assay may open up a new perspective for designing promising phosphorescent materials for biosensor.