A method of detecting a preload of a linear guide includes: applying an external force to the linear guide with an external force applying device, wherein the external force applying device sends an impact signal while applying the external force; sensing with a sensor a vibration signal sent from the linear guide because of vibration thereof which occurs under the external force; and receiving the impact signal of the external force applying device and the vibration signal of the sensor and calculating the preload of the linear guide according to a received result, with a signal analyzer. Therefore, with the method of the present invention, the preload of the linear guide is precisely tested regardless of environmental factors.