The present disclosure provides a semiconductor structure and a manufacturing method thereof. The manufacturing method of a semiconductor structure includes providing a substrate having trenches, regions other than the trenches in the substrate form a plurality of active regions at intervals; forming a first isolation layer and a second isolation layer, a top surface of the first isolation layer is lower than a top surface of the second isolation layer, a groove is formed between the second isolation layer and the active region; forming a barrier layer in the groove, an etching rate of the barrier layer is lower than an etching rate of the first isolation layer; and forming a third isolation layer in an intermediate trench, the intermediate trench is filled with the third isolation layer, and the first isolation layer, the second isolation layer, the third isolation layer, and the barrier layer form an isolation structure.