A re-distribution layer structure is adapted to be disposed on a substrate having a pad and a protective layer which has a first opening exposing a part of the pad. The re-distribution layer structure includes a first and a second patterned insulating layers and a re-distribution layer. The first patterned insulating layer is disposed on the protective layer and includes at least one protrusion and a second opening corresponding to the first opening. The re-distribution layer is disposed on the first patterned insulating layer and includes a pad portion and a wire portion. The pad portion is located on the first patterned insulating layer. The wire portion includes a body and at least one trench caved in the body. The body extends from the pad portion to the pad exposed by the first and the second openings. The body covers the protrusion, and the at least one protrusion extends into the at least one trench. The second patterned insulating layer covers the wire portion and exposes a part of the pad portion. A manufacturing method of re-distribution layer structure is further provided.