Amphiphilic poly(amidoamine) (PAMAM) dendrimers are a well-known dendritic family due to their remarkable ability to self-assemble on solid surface. However, the relationship between molecular conformation (or adsorption kinetics) of a self-assembled layer and molecular amphiphilicity of such kind of dendrimer is still lacking, which limits the development of modulating self-assembling structures and surface functionality. With this in mind, we synthesized a series of amphiphilic PAMAM-based dendrimers, denoted as G(1)C(n), with different alkyl chains (n = 8, 12, and 16), and investigated the molecular aggregation on silica surfaces by means of quartz crystal microbalance with dissipation, atomic force microscopy, and contact angle. After rinsing, remaining adsorption amounts of G(1)C(12 )were higher than those of G(1)C(8) at high concentrations, suggesting that G(1)C(12) adlayers were more stable due to the stronger intermolecular hydrophobic interactions, whereas it preferred to adopt the intramolecular hydrophobic interactions for G(1)C(16), with low adsorption amounts and unstable adlayers. Bilayer-like structures were inferred in G(1)C(8) and G(1)C(12) adlayers with loose conformation, whereas monolayer structures were likely to exist in the sparse adsorption film of G(1)C(16). Our results provided more detailed understanding of the effect of molecular structure on the self-assembled structures of amphiphilic dendrimers on solid surfaces, shedding light on the controlled microstructure and wettability of functional surface by modulating the length of hydrophobic chains of dendrimers and a potential application of dendrimer-substrate combinations.