Among the diverse sensing techniques, fluorimetric detection dominates over the other methods because of its rapid signaling, high selectivity and sensitivity, and operational simplicity. This present article delineates fabrication of a fluorescent organic nanoparticle-protamine (FONP-Pro) conjugate for selective and sensitive detection of heparin simply by exploitation of the aggregation-induced emission (AIE) property of the FONPs. Naphthalene diimide-based bola-type amphiphilic molecules (NDI-1) comprise a naphthyl residue and a 3-aminopyridyl unit at both terminals, forming organic nanoparticles in a dimethyl sulfoxide-water binary solvent mixture, and exhibited AIE through excimer formation. The presence of naphthyl residue in the molecular backbone facilitates the intramolecular charge transfer to generate orange-emitting (λem = 594 nm) AIE-luminogen (AIE-gen). The aminopyridine residues within NDI-1 induced negative surface charge on NDI-1 FONPs, which facilitated interaction with positively charged protamine (Pro) to construct FONP-Pro conjugates. Formation of this NDI-1 FONP-Pro conjugate through the interaction between Pro and FONP drastically reduced the orange emission intensity (fluorescence off) of the AIE-gens. Interestingly, addition of heparin to this FONP-Pro conjugate turned on the fluorescence signal of FONPs through unwinding of the Pro from the FONP surface because of a strong binding affinity between heparin and Pro. Formation of the FONP-Pro conjugate and fluorimetric sensing of heparin was investigated by monitoring the change in emission behavior of NDI-1 FONPs. Also, the heparin-sensing was found to be highly selective against many other biomolecules including proteins, enzymes, and DNA. Hence, a selective and efficient heparin sensor (FONP-Pro) was developed having a limit of detection of 12 nM simply by utilizing the fluorescence "turn-off" and "turn-on" mechanism of NDI-1 FONP.