A flexible substrate is provided, and the flexible substrate includes a second organic layer, a first inorganic layer, a first organic layer, and a second inorganic layer, which are sequentially stacked. A surface, which is in contact with the first organic layer, of the first inorganic layer has a first groove. A surface, which is in contact with the first inorganic layer, of the second organic layer has a second groove. A surface, which is in contact with the second inorganic layer, of the first organic layer has a third groove. In a direction perpendicular to the first inorganic layer, any two of the first groove, the second groove and the third groove at least partially are not aligned with each other. A manufacturing method of the flexible substrate, a panel including the flexible substrate, and an electronic device including the flexible substrate are also provided.