The invention relates to a natural gas neon isotope composition measuring device. The device comprises a rare gas mass spectrometer, active carbon cold traps I and II and a natural gas steel cylinder,wherein the natural gas steel cylinder is connected with a high vacuum system through the active carbon cold trap I through a high vacuum pipeline; the high vacuum pipeline is connected with an ultra-high vacuum pipeline through a pipeline, and the ultra-high vacuum pipeline is connected with the active carbon cold trap II, the rare gas mass spectrometer and an ultra-high vacuum system; a pressure reducing valve, high vacuum valves I and II and a film vacuum gauge are arranged between the natural gas steel cylinder and the active carbon cold trap I; the active carbon cold trap I is connectedwith the high vacuum pipeline through a high vacuum valve III; a high vacuum valve IV is arranged between the high vacuum system and the pipeline; an ultra-high vacuum valve I is arranged on the pipeline; an ultra-high vacuum valve II is arranged between the ultra-high vacuum system and the pipeline; the active carbon cold trap II is connected with an ultra-high vacuum pipeline through an ultra-high vacuum valve III; and an ultra-high vacuum valve IV is arranged between the rare gas mass spectrometer and the active carbon cold trap II. The invention also discloses a using method of the device.The device has the advantages of accurate measurement result and no interference.