We report a simple, fast, sensitive and label-free fluorescent assay for sensing heparin based on the electrostatic attraction between heparin and coralyne. Under optimal conditions, the sensor exhibits high sensitivity, and needs just 10 min to complete the detection process. Additionally, the proposed sensor is simple to operate and low cost with no chemical labeling or modification of DNA required. Finally, the constructed sensor can be applied in Tris–HCl buffers and also complicated human serum samples.