The DNA hairpin with a limited size is sensitive to temperature. However, it is unclear if thermosensitive TRP channels also use the temperature-dependent little hairpin sizes to govern their temperature thresholds and sensitivity. Here, graph theory was used as a novel tool 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 biggest little hairpin with the minimum topological loop size and strength control the temperature threshold while a change in the total minimum little hairpin sizes upon a change in the total non-covalent interactions along the channel gating pathway governs the temperature sensitivity. This size- and strength-dependent hairpin thermodynamics may tune the thermal activity and sensitivity of biological macromolecules.