The instability of the shear layer separated from a circular cylinder is studied with the Reynolds number (Re) of 3000～104 by numerically solving the two-dimensional Navier-Stokes equations. In the wake of the cylinder, primary vortex shedding with natural frequency fs occurs, and the instability of the shear layer with frequency ft develops, which leads to mixing layer eddies and interacts with the primary shedding vortices. However, there remains some uncertainties regarding to the variation of the shear layer characteristic frequency with the Reynolds number. Based on the previous experimental work, several relationships of ft/fs with Re has been proposed including ft/fs～Re0.5 by Bloor, ft/fs～Re0.87 by Wei and Smith and ft/fs～Re0.67 by Prasad and Williamson. The objective of this study is to predict reasonably the relation of the shear layer instability frequency with the Reynolds number based on the present accurate calculation with the high-order schemes and high-resolution spectrum analysis. According to our calculated results, a variation for the normalized shear-layer frequency of the form ft/fs～Re0.69 is predicted numerically, which is in good agreement with a recent experimental measurement of Re0.67 and physical prediction of Re0.7.