Micro cantilevers in atomic force microscopy are important force sensors in nano research, and the spring constant is one of the most important parameters of the cantilevers. Normal testing methods are not suitable for the spring constant detecting of micro cantilevers according to the strict scale of the cantilevers, and new methods are needed to the study of micro cantilevers. A method for detecting of spring constant of micro cantilevers based on combining the numerical simulation and frequency measurements is presented in this paper. The new method involves four steps, the first step is developing the vibration model of the micro cantilever studied immersed in air and determine the fluid parameters in the model during dynamic tests in atomic force microscopy presented in this paper; the second step is analyzing the vibration behavior of the corresponding cantilevers with the same geometry but different young’s modulus. The third step is measuring the natural frequencies of the micro cantilevers and comparing the experimental results with the numerical results to determine the young’s modulus of the cantilever. The last step is conducting the young’s modulus to the cantilever FEA model for determination of its spring constant. Experiments on a NSC cantilever have been done to validate the method presented in this paper.