It is demonstrated that the characteristic matrix method is effective and reliable for the optical identification of two-dimensional layered nanomaterials on different substrates. By using this method, the authors calculate the reflectivity and optical contrast of layered MoS2 crystallites prepared on quartz by chemical vapor deposition. It is found that the measured pixel intensity of an MoS2 optical image under continuous spectrum light is proportional to the calculated reflectivity, and that the theoretical optical contrast agrees well with the experimental results. This work provides a new way for the calculation of the optical contrast of the 2D nanomaterials and layered heterostructures on various substrates.