In this work, we report an approach to prepare segmented polymer nanofibers (SPNFs) composed of rodlike subunits by kinetically controlled self-assembly of polystyrene-b-poly(4-vinylpyridine)-based supramolecules in microfluidic chips. The length and morphology of the SPNFs could be effectively adjusted by changing the total flow rate (V-total) and the molar ratio (x) of 4-vinylpyridine (4VP) unit to a hydrogen-bonding molecule, 3-n-pentadecyphenol. Moreover, the subunits of SPNFs could transform from short rods to spheres when the interfacial tension between PS core and solvent increased. On the contrary, the SPNFs elongated along the major axis when the interfacial tension decreased. This work not only offers mechanism insights into the hierarchical self-assembly of block copolymer-based supramolecules but also provides a versatile and effective method for kinetically controlling the hierarchical structures of assemblies.