Sulfide (sulfur ether or thioether) is a kind of chemically active compound in petroleum, which has special significance in both geochemistry and oil processing. However, the molecular characterization of sulfides is a challenge because the composition of sulfides is very complex and each individual sulfide generally has a very low concentration. Separation is necessary for an in-depth characterization of these compounds, but it is not easy to achieve thus far. Here, we developed a method for the isolation of sulfides from petroleum fractions. Sulfides have different adsorption behaviors from the petroleum matrix on the aluminum oxide and silica gel. The petroleum fraction was first separated by an alumina column to obtain the mixture of sulfides and nonpolar hydrocarbons, and then the mixture was transferred into the second column, which was packed with silica gel. Hydrocarbons were removed by a nonpolar solvent, and sulfides were eluted from the column with a polar solvent. Isolated fractions were characterized by high-resolution mass spectrometry and gas chromatography (GC). Results show that the method is simple and practical. The purity of the sulfide fraction is acceptable for composition analysis by GC-relevant techniques. The method could be used for routine analysis of sulfides in crude oil and its products.