Supramolecular assembly to form a large variety of nanostructures has received increasing attention for diverse applications, in particular biomedical applications involving drug delivery, bioimaging, therapy and regenerative medicine. Meanwhile, the modulation of morphology and structure of nanoassemblies is a still big challenge. Herein, we report a series of supramolecular structures (BP-KLVFFG-PEG, BKP) and elucidate that their morphological transformation process is modulated by H-bonding, π-π interactions and hydrophilic/lipophilic balance (HLB). Our studies reveal that the hydrophobic and π-π interactions initially drive the self-assembly of BKP into nanoparticles in J-type aggregates in water, and the H-bonding interactions further induce an in situ spontaneous morphology transformation into nanofibers. The conversion rate is related to the length of the hydrophilic chains. The nanofibers are maintained by β-sheet H-bonds with parallel structure. Our results provide insight into the relationship between molecular structures and morphological transformations of self-assembled nanomaterials, which will guide the design of complex self-assembled materials in biological conditions.