Source-specific biomarkers are the best targets for isotope analysis and paleoclimatic applications. Compound-specific hydrogen isotope (δ²H) measurements of sterols can be applied to the reconstruction of hydroclimatic conditions and paleo-sea surface salinity. Precise measurements using gas chromatography-isotope ratio mass spectrometry require a high baseline resolution between target analyte and adjacent compounds. However, isotope analysis of sterols for paleoclimatic applications is limited due to the difficulty in purifying complex natural samples. This study aimed to review several improved purification methods of source-specific sterols using liquid chromatography and the applications of compound-specific isotope analysis of sterols in lakes or seas. In addition, we successfully purified dinosterol and brassicasterol from the Chinese marginal sea surface sediments, meeting the requirements for δ²H and carbon isotope analysis. This study provides significant insights and technical support for the paleoenvironmental reconstruction in the Chinese marginal seas.