A new calibration method for exact measurement of friction force in atomic force microscope (AFM) is presented. A new conversion factor involves a contact factor affected by tip, cantilever and contact stiffness. Especially the effect of contact stiffness on the conversion factor between lateral force and lateral signal is considered. Conventional conversion factor and a new modified conversion factor were experimentally compared. Results showed that a new calibration method could minimize the effect of normal load on friction force and improve the conventional method. A new method could be applied to the specimens with different physical properties.