A two-step chromatographic process was developed for the fractionation of lignosulfonate (LS), a main component of sulfite effluence. LS fractions with low polydispersity were obtained, which enables detailed investigation into the relationship between structure and property of LS with different molecular weight (M-w). Sephacryl S-100 was used for LS separation in the first step, and then Sephadex LH-20 and Sephacryl S-100 were chosen for further separation of LS fractions with M-w below and above 6000 Da, respectively. Although the mechanism of column chromatographic separation of LS was mainly dependent on its molecular size, 0.20 mol/L of NaCl or NaNO3 solution was needed in the mobile phase at pH 5.7 to minimize electrostatic interaction and polyelectrolyte effect. Compared with the polyelectrolyte effect, the electrostatic interaction was more important in the LS separation. Because the LS fractions of higher M-w had a characteristic of low negative charge, a mobile phase with high flow rate and high pH value was suitable for separating LS fractions with high M-w.