Hyperbranched polymers (polyesteramides) were used as a cationic fixing agent to remove dissolved and colloidal substances (DCS) from the water phase in a fiber suspension. The relative turbidity, electrophoretic mobility, and average diameter of the colloidal particles were determined as a function of polyelectrolyte concentration. The results indicated that maximum removal of DCS was achieved at about zero electrophoretic mobility of the suspension where the negative surface charges of particles were neutralized by the oppositely charged hyperbranched polymer. The amount of hyperbranched polymers needed to maximize DCS fixation on the fibers was higher than the amount of poly-DADMAC (diallyldimethylammonium chloride) needed to reach the same effect. This was found to be due to the lower molecular weight and lower charge density of hyperbranched polymers. The lower molecular mass allowed penetration of these polymers into pores of fibers that resulted in higher polymer consumption before removal of the dissolved and colloidal substances from the fiber suspensions. A lower charge density further resulted in a higher saturation adsorption of the hyperbranched polymer. Experiments with both DCS and model-latex particles showed that the initial increase in relative turbidity was due to the aggregation of particles before fixation to fibers. The results from quartz crystal microbalance with dissipation (QCM-D) experiments showed that the efficiency of hyperbranched polymer and poly-DADMAC was similar even if they had different structure. Therefore, this study highlights the importance of molecular mass and charge density of the polymers as well as the surface structure of polymer layers which in turn gives implications for development of new structures of fixing agents.