In the papermaking process, the removal and control of dissolved and colloidal substances (DCS) is a key issue for reducing the usage of fresh water. The use of nano TiO2 for removal of dissolved substances (DS) and colloidal substances (CS) was investigated through monitoring the titration process of nano TiO2 colloids to sodium laurate (C11H23COONa, DS simulacra) and stearic acid (C17H35COOH, CS simulacra) solution with COD (chemical oxygen demand), DLS (dynamic light scattering), SEM (scanning electron microscope), and zeta potential, respectively. The results indicated that most of the simulacra molecules could be removed from the aqueous solution by the flocculation with nano TiO2 colloids. The removal of CS by nano TiO2 colloid arose from heterocoagulation rather than from charge neutralization, in which nano TiO2 was adsorbed onto the surface of CS particles and bridged CS to form flocs. While in the removal process of DS by nano TiO2, the negative-charged portion of the DS molecule was adsorbed onto the surface of nano TiO2 particles with a mono- or multilayer, eliminating the repulsive force between nano TiO2 particles and resulting in their flocculation.