We have developed a ratiometric sensor based on polyelectrolyte-induced pyrene excimers for the rapid and visual detection of heparin in aqueous media. This sensor has a simple preparation method, fast response, low cost, and high sensitivity and selectivity. As a complementary strategy for the detection of heparin based on the formation of pyrene excimers, this approach could identify heparin specifically over chondroitin sulphate, which is the major interferent in most sensing systems for the detection of heparin. The detection limit of this approach can be as low as 0.14 μM using fluorescence measurements. To the best of our knowledge, this is among the lowest LOD for the detection of heparin reported in the literature. It enables the rapid and visual detection of heparin both in buffered solution and serum samples.