A positively charged aggregation-induced emission luminogen (AIEgen), TPE-P+, was constructed by linking a pyridyl cation to tetraphenylethylene (TPE) via a cyanoethylene bond. TPE-P+ can realize the identification of heparin (Hep) by aggregating with negatively charged Hep via electrostatic interactions. Upon addition of Hep, TPE-P+ exhibited 36-fold fluorescence enhancement in less than 5 s, exhibiting quick and sensitive response to Hep with a low detection limit down to 4 nM. Among various biological substances, even Hep analogs like chondroitin 4-sulfate and hyaluronic acid, TPE-P+ showed the most significant fluorescence enhancement to Hep only, demonstrating its excellent selectivity for Hep. In particular, with long-wavelength emission near 600 nm and large stocks shift (∼160 nm), TPE-P+ enabled minimization of autofluorescence interference from a complex biological matrix and provided more accurate results. Finally, TPE-P+ was successfully applied for sensitive and selective detection of Hep in serum. Notably, there existed a good linear relationship in a serum assay when the Hep concentration ranging from 0 to 4 μM (R2 = 0.9934) covered the clinical dosage level during both cardiovascular surgery and long-term care, suggesting the potential clinical practice for quantifying Hep in serum. Moreover, TPE-P+-Hep complex can be further disaggregated by protamine (PRTM) due to the stronger affinity between Hep and PRTM, thereby leading to further detection of PRTM effectively. Last, but not least, the "off-on-off" system designed for both Hep and PRTM detection proved to be reversible.