Abstract: In hydrogen isotope ratio analysis of single molecule hydrocarbons by gas chromatography-high temperature thermal conversion-isotope ratio mass spectrometry, different split ratios (0.1, 0.5, 1.0, 2.0, 5.0 and 10.0)were selected to study the corresponding hydrogen isotope ratios. The results showed that different inlet split ratios influenced in various extents on the peak area and peak width, namely, the peak area and peak width decreased exponentially with the increase of split ratios. Especially when split ratios were less than 1.0, the uncertainty of the hydrogen isotope determination results increased. And when the split ratios were relatively larger, hydrocarbon isotope determination results became stable. The reasons for this phenomenon were also discussed. The research results also showed that in the hydrogen isotope analysis, the choice of a larger split ratio did not lead to significant isotopic fractionation and was more favorable for the sample analysis.