The present disclosure provides a manufacturing method of a semiconductor structure and a semiconductor structure. The manufacturing method of a semiconductor structure includes: providing a substrate, the substrate includes active regions and isolation regions, each of the isolation regions includes a first trench and an isolation layer formed in the first trench; removing part of the isolation layer to form first grooves; forming a first mask layer, the first mask layer covers upper surfaces of the active regions and fills the first grooves; planarizing the first mask layer, such that an upper surface of a portion of the first mask layer located above the active regions is flush with an upper surface of a portion of the first mask layer located above the isolation regions; removing part of the first mask layer, part of the isolation layer, and part of the substrate, to form second trenches and third trenches.