Two-dimensional (2D) materials, with remarkably electronic, optical, and thermal properties, have attracted tremendous attention in recent years. To meet the rapid development of 2D materials, it’s fundamentally necessary to the precisely characterize the structure and properties of 2D materials. Raman spectroscopy has been proven to be a fast, convenient, and nondestructive technique to characterize the basic structural information of low dimensional materials. In this review, we introduce recent advances in Raman spectroscopy for structural characterization of 2D materials. We mainly focus on the characterization of layer number, stacking order, crystalline orientation, defects, and structural phase transition of 2D materials by Raman spectroscopy.