Heparin (Hep) is a highly negatively charged linear glycosaminoglycan involved in various physiological processes, especially blood coagulation. Hep is also a first-line drug for anticoagulation and prevention of thromboembolism, but its overdose will cause serious side effects. Herein, we designed a long-wavelength double-charged cationic fluorescent probe PYPN, and studied its aggregation state and detection performance for Hep. PYPN was readily synthesized through a one-step reaction without complicated purification. In aqueous medium, PYPN molecules with an amphiphilic structure spontaneously form nano-assemblies, which can be immediately decomposed by Hep due to the formation of a PYPN-Hep complex based on electrostatic attraction. The assembly shows a fast, sensitive and ratiometric fluorescence response to Hep, without being obviously interfered by other compounds. In various serum matrices, the fluorescence intensity ratio F610/F470 has a good linearity with Hep concentration (0-12 μg mL-1), and the detection limit (0.11-0.12 U mL-1) is lower than the minimum concentration (0.2 U mL-1) used in clinical treatment. Our study provides an easy-to-prepare and feasible tool for the selective and sensitive quantification of Hep in serum.