It is well known that the membrane separation process combined with surfactant micelle (micellar-enhanced ultrafiltration) or polyelectrolyte (polyelectrolyte-enhanced ultrafiltration) can remove heavy metal ions or radionuclides effectively. However, the complexing agent, surfactant or polyelectrolyte remained in effluent is a serious disadvantage of these methods. In this study, humic substances (HS) were used as complexing agents instead of synthetic chemicals. The HS are sorts of natural organic matters and their functional groups such as carboxyl and phenyl groups can bind with the cation and form complexes. The effects of HS concentration and pH on the removal of cobalt were investigated. At the HS concentration of 3g/L and pH of 6, over 95% of cobalt was removed by regenerated cellulose membrane with molecular weight cut-off (MWCO) of 3000. As the HS concentration increased, the removal of cobalt was also enhanced because of the increase in binding sites (functional groups). The removal of cobalt increased from 72.5% to 97.5% as pH increased from 4 to 8 at the HS concentration of 3g/L. It resulted from the more deprotonation of functional groups in humic acid at higher pH.