Recently, low molecular weight gelators are getting increasing interests in pharmaceutical field. In the current study, puerarin (PUE), a natural and bioactive flavonoid from the plant Pueraria lobata, was found to be able to gelate water to form hydrogel during cooling down process of its hot solution. In order to explore its gelation mechanism, the crystallization process, characterization, interaction force, morphology, rheological properties of PUE hydrogel were investigated by polarized light microscopy (PLM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), Karl Fischer (KF), Raman spectrum, 1H-NMR, environmental scanning electron microscope (ESEM) and rheometer. The results indicated that at a high supersaturation condition, the gelation process of PUE was initiated by nucleation, and then fibers emanated rapidly from the nucleation center. The types of fiber growth changed from “fiber-like” growth to “less-linear” growth as the increase of supersaturation, leading to a denser structure with higher fractal dimension and stronger mechanical properties. Notably, the C=O…π interaction facilitated the gelation assembly process, and the crystallographic mismatch branching played a vital role in constructing the network of PUE hydrogel.