A method of manufacturing a semiconductor structure includes: receiving a substrate having an active region and a non-active region adjacent to the active region; forming an etch stop layer over the non-active region of the substrate, in which the etch stop layer is oxide-free; forming an isolation over the etch stop layer; removing a portion of the active region and a portion of the isolation to form a first trench in the active region and a second trench over the etch stop layer, respectively, in which a thickness of the etch stop layer beneath the second trench is greater than a depth difference between the first trench and the second trench; forming a dielectric layer in the first trench; and filling a conductive material on the dielectric layer in the first trench and in the second trench. A semiconductor structure is also provided.