The invention relates to an quantitative determination method of the isotope of hydrogen, hydrogen deuteride and hydrogen deuterium, which comprises using neon gas to be carrier gas, leading a gas chromatograph to adopt a double-strut double-temperature system, choosing 50 DEG C sampling temperature, sampling through an injector or a gas sampling valve, and the sampling quantity is 0.1-2.2mL, sample gas which is separated by a gas-chromatography column generates electrical signals through a detector, recording the pear areas of hydrogen, hydrogen deuteride and deuterium gas of chromatogram peaks through a chromatogram working station after the electrical signals are magnified through a front magnifier, adopting a volume correction factor normalization method or an external standard method to measure the content of hydrogen (H2), hydrogen deuteride (HD) and deuterium (D2), and getting detection results. The invention has the advantages of excellent separation effect of hydrogen isotope and high detection sensitivity, the detection of the pureness of deuterium gas can reach more than 99.99%, hydrogen deuteride (HD) and deuterium (D2) are sampled in big dosage, the peaks are not overlapped, thereby guaranteeing the quantitative detection of high-purity deuterium gas, and standard gas is not needed to be prepared in the process of detecting. The method simplifies the operation process, which is convenient for on-line detection of deuterium gas in the process of producing.