The present disclosure provides a micro-fluidic substrate, a micro-fluidic structure and a driving method thereof. The micro-fluidic substrate of the preset disclosure includes a substrate, and a plurality of driving electrodes on the substrate and configured to drive a droplet to move, the plurality of driving electrodes being in a same layer with a gap space between adjacent driving electrodes. The micro-fluidic substrate further includes: at least one auxiliary electrode on the substrate and configured to drive the droplet to move, an orthographic projection of the auxiliary electrode on the substrate at least partially overlapping with an orthographic projection of the gap space on the substrate, and the auxiliary electrode and the driving electrodes being in different layers.