The invention relates to a micro-fluidic chip for inhibiting electroosmotic flows through grafting a polyelectrolyte brush on the surface of a micro-channel, belonging to the field of microfluidics. The micro-fluidic chip comprises a sample feeding pool, an inlet channel, a slit channel, an outlet channel and a waste liquid pool which are sequentially communicated, wherein a polyelectrolyte brush with cations is grafted on the channel surface of the inside of the slit channel, and an uniform electric field is applied parallel to the direction of the channel so as to drive fluid to flow. Under the action of the uniform electric field, the polyelectrolyte brush grafted on the surface of the micro-channel extends along the direction of the electric field, and the fluid in the micro-channel is under the action of the viscous friction and electrostatic attraction of the polyelectrolyte brush, so that surface charges in the channel are balanced, reduced or inhibited; and due to the attraction action among solvent particles and the polyelectrolyte brush, the solvent particles are impregnated into a polyelectrolyte brush layer, thereby achieving the purpose of inhibiting electroosmotic flows. The micro-fluidic chip disclosed by the invention can improve the separation precision of proteins and DNA (deoxyribonucleic acid) molecules, and has the advantages of wide applicability, strong controllability and the like.