Petroleum sulfonates are widely used in chemical flooding in China. The samples are complicated and not all the fractions are effective. To recognize effective fraction with excellent interfacial activity and emulsifying property, a preparative liquid chromatography technology was adopted to perform the fraction separation for petroleum sulfonate. Structural composition, interfacial activity，emulsifying property and flooding efficiency of obtained fractions were also analyzed and discussed. It was shown that petroleum sulfonate can be effectively separated into different fractions with obvious structural differences, among which one fraction with the most excellent interfacial activity and the emulsifying property was recognized. The average molecular weight and molecular weight distribution range of this active fraction were 455.5 and 428–468, respectively, which are both crucial for activities. Alkylbenzene dicyclohexane sulfonates, alkyl indan sulfonates, and alkyl naphthalene sulfonates are major structure types, and alkyl chain length is mainly distributed between 12–15, 15–17 and 15–17 respectively. Besides, this active fraction is universal, and it can produce ultralow interfacial tension (~10 3 mN/m) and good emulsifying property against different crude oils including Xinjiang, Changqing, Liaohe Oilfields, etc. in China. The developed technology herein can be used to track and confirm the characteristics and/or active fraction in petroleum sulfonate accurately.