When a traditional Atomic Force Microscope (AFM) is used to measure the critical dimension (CD) of a line, the line width and the shape of two side-walls are difficult to be measured due to the probe shape and positioning. Therefore, this paper proposes a AFM with dual probes to eliminate the effect of probe shapes on measuring results. A alignment system based on machine vision for the AFM with dual probes was introduced, the system contacted the two probes each other to implement the alignment of the two probes in 3D directions. A lens with sub-micron resolution was used in the system to match a high resolution CCD to obtain the clear images and to acquire the movement states of two probes in both horizontal and vertical directions. Furthermore, the novel self-sensing and self-actuating probe based on a quartz tuning fork combined was used for dynamic mode AFM to simplify the system, by which it not needed the external optical detection system, and its dimension was shortened and the affect come from stray light was eliminated. Finally, by extracting sub pixel edge of the tips, the related position between two probes was obtained accurately and the alignment of two probes in sub micron(within 1 μm)was implemented. The results were verified by the distance between two probes and amplitude/phase curves.