To explore the thermal-responsive characteristics of acetylated amylose-guest V-type helical complexes (AAGHCs) and their potential use as thermal-responsive drug carriers, different types of AAGHCs were built, in which acetylated amylose was used as a host, and iodine, propofol, or hexane was utilized as the guest molecule. Their thermal-responsive characteristics were investigated through molecular dynamic (MD) simulation and corresponding experiments. MD simulation showed that the thermal-responsive helix-unfolding and guest-release behavior in AAGHCs, and the complete unfolding of AAGHC could be divided into brewing, triggering and collapsing periods. Energy analysis revealed that the Lana-Jones potential is an important binding energy that bridges host and guest molecules and enhances the stability of the helix. The various types or number of guests showed different binding energies. The stronger the binding energy, higher is the temperature required to trigger the unfolding of the helix and the releasing of guests. FT-IR and X-ray diffraction analyses confirmed the structures of AAGHCs. The change in hydrated size, and UV-VIS absorption of AAGHCs at high temperatures both confirmed the thermal-responsiveness of AAGHCs. The fluorescence fluctuation of loaded 7-hydroxycoumarin reflected the same thermal-responsive process and mechanism as MD simulation. This study provides meaningful theoretical guidance for the design of thermal-responsive drug carriers based on acetylated amylose-guest V-type helical complexes.