The DNA hairpin with a limited size is sensitive to temperature. However, it is unclear if thermosensitive TRP channels also use temperature-dependent hairpin sizes to govern their activation temperature thresholds and sensitivity. Here, graph theory was used as a novel method to test this hypothesis by analyzing the redox- and state-dependent cryo-electron microscopy structures of TRPV3 with or without the Y564A mutation at different temperatures. The results showed that the largest hairpin topological loop size and a change in total hairpin sizes along the channel gating pathway control the temperature threshold and sensitivity, respectively. This size-dependent hairpin thermodynamics may mediate the thermal stability and activity of biological macromolecules.