Any of two or more two-dimensional (2D) materials with similar properties can be alloyed into a new layered material, namely, 2D alloy. Individual monolayer in 2D alloys are kept together by Van der Waals interactions. The property of multilayer alloys is a function of their layer number. Here, we studied the shear (C) and layerbreathing (LB) modes of Mo 0.5 W 0.5 S 2 alloy flakes and their link to the layer number of alloy flakes. The study reveals that the disorder effect is absent in the C and LB modes of 2D alloys, and the monatomic chain model can be used to estimate the frequencies of the C and LB modes. We demonstrated how to use the C and LB mode frequency to identify the layer number of alloy flakes deposited on different substrates. This technique is independent of the substrate, stoichiometry, monolayer thickness and complex refractive index of 2D materials, offering a robust and substrate-free approach for layer-number identification of 2D materials. [6][7][8][9] The 2D alloys are a rich source of 2D materials because they can exhibit tunable properties because the ratio of two end compositions can be controllable. 6,9 The key to form a good quality of 2D monolayer is mixing the end compositions at atomic scale. Individual monolayers in the 2D alloys are kept together by Van der Waals interations. Similar to graphite and graphene, the property of multilayer alloys is a function of their layer number. How to determine the layer number of ultrathin 2D alloys is thus of primary importance for fundamental science and applications.Several optical techniques have been developed to identify the layer number of 2D materials. [10][11][12][13][14][15][16] Nevertheless, most techniques rely on the specific properties of 2D materials. For example, one can identify the layer number, denoted as N, of MoS 2 by the intensity or peak positions of the corresponding photoluminescence (PL) peak and Raman modes. 14,15 The mostly used technique is based upon optical contrast of 2D materials against dielectric substrates, such as a Si substrate covered with a SiO 2 layers. 10,12 To precisely identify N of few-layer 2D materials, the experimental optical contrast must be compared with the theoretical one for different N. However, the theoretical calculations require predetermined parameters, including thickness of a single layer, complex refractive index of the material and information of the substrate, 11,17 which poses great challenge for research on new 2D materials in early stage. Therefore, it is desirable to develop a novel approach to identify the layer number of fewlayer 2D materials, relying on as little prerequisite parameters as possible.The interlayer modes of multilayer 2D materials originates from the relative motions of the rigid monolayer planes themselves, either perpendicular or parallel to their normal, such as the shear (C) modes and the layer breathing (LB) modes. [18][19][20][21][22][23][24] The C and LB modes are the collective vibration modes of all the layers so their frequency (ω C and ω LB ) depe...