The rational design of perfluorinated amphiphiles to control the supramolecular aggregation in aqueous medium is still a key challenge for the engineering of supramolecular architectures. Here we present the synthesis and physical properties of six novel non-ionic amphiphiles. We also studied the effect of mixed alkylated and perfluorinated segments in a single amphiphile and compared it with only alkylated and perfluorinated units. To explore their morphological behavior in aqueous medium, we used dynamic light scattering (DLS) and cryo-TEM/EM measurements. We further confirmed their assembly mechanisms with theoretical investigations, using the Martini model to perform large-scale coarse-grained molecular dynamics simulations. These novel synthesized amphiphiles offer a greater and more systematic understanding of how perfluorinated systems assemble in aqueous medium and suggest new directions for rational designing of new amphiphilic systems and interpreting their assembly process.