An interconnection structure and a manufacturing method thereof are provided. The method includes the following steps. First, a substrate having a first surface and a second surface opposite to each other is provided. Then, a conductive through via extended from the first surface to the second surface is formed in the substrate. Then, a portion of the substrate is removed from the first surface to expose a portion of the conductive through via. Then, a dielectric layer is formed on the substrate, and the dielectric layer covers the exposed conductive through via. Then, an opening is formed in the dielectric layer, wherein the opening exposes a portion of the conductive through via, and the top surface of the conductive through via protrudes from the bottom surface of the opening. Then, a conductive layer is formed in the opening.