In order to investigate the supramolecular assembly and intermolecular hydrogen bonding of special amphiphiles, two bolaform amphiphilic Schiff bases (GN1 and GN2) with different hydrophilic spacers were designed, and their supramolecular assembly and interaction properties were investigated by spectral measurements. It was found that both the Schiff bases showed hydrogen bonding interaction with barbituric acid. In addition, it was interesting to note that the spacer groups of the molecules had effect on the molecular ratio in the hydrogen-bonded complexes. Due to the directionality and strong matching of hydrogen bond, one barbituric acid molecule can be encapsulated into the intramolecular area of GN1, while two barbituric acid molecules were trapped into the GN2 molecule through intermolecular H-bonds for GN2 due to the long spacer and flexible structure. A rational complex mode was proposed.