The DNA hairpin with a limited size or strength is sensitive to temperature. However, it is unclear if thermosensitive TRP channels also use the temperature-dependent little hairpin sizes or strengths 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 mouse TRPV3 with or without the Y564A mutation at different temperatures. The results showed that the biggest little hairpin with the minimal topological loop size and strength determines the temperature threshold while a change in the total minimal little hairpin sizes upon a change in the total little hairpin-dependent 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.