The host-guest chemistry of dendrimer-biomacromolecule complexes is of great significance to both design and optimization of dendrimer-based drug delivery and host-guest systems. Here, we characterized the interactions between dendrimer and heparin by isothermal titration calorimetry (ITC), H-1 nuclear magnetic resonance (1H NMR), pulsed-field gradient (PFG) NMR, nuclear Overhauser effect spectroscopy (NOESY), and atomic force microscopy (AFM) studies. The calorimetric results suggest that miscellaneous aggregates are formed at different stages when heparin was titrated into a dendrimer solution: dendrimer-heparin necklace structures, followed by the formation of larger and more stable aggregates, and then macroscopic complexes which precipitate from the solution. The binding process is significantly influenced by dendrimer generation, surface functionality, and ion strength, indicating that the formation of dendrimer-heparin aggregates is predominantly driven by electrostatic interactions. The NMR results confirm the dendrimer-heparin binding models established by calorimetric measurement and present a new type of dendrimer-heparin aggregates at higher heparin/dendrimer molar ratios. Formulations containing generation 5 (G5) PAMAM dendrimers with a heparin/G5 molar ratio of 0.5-1.2 are proposed as effective ones for the treatment of thrombosis in noninvasive delivery routes such as nasal, pulmonary, transdermal, and oral routes. The combination of ITC and NMR in this study provides new insight into the interactions between globular and linear polymers and the delivery of macromolecular therapeutics such as heparin by dendrimers.