In the high precision image measurement system, the verticality error between the axis of the shooting system and the measured object can bring error of the measurement result. The high demand of the system’s vertical degree is raised by measure system due to the demands of high precision and disposable full field imaging in the micro-parts imaging measurement. The existing method of optical axis verticality detection cannot meet the demand all. In order to achieve the high-precision adjustment of the system optical axis, the algorithm of detecting verticality based on regional image definition is proposed. First, the objected standard image is divided into fixed area. Then, the object plane is moved from the downside to the upside of the focus plane, meanwhile, recording the definition function values of each standard image region at each step, and fitting out the clearest positions of the regions. Finally, according to the inter-regional relations between the locations and the height difference of the each regional clearest position, the small angle between the optical axis and the measured surface can be calculated. The experiment is based on the given image of lithography template with the scale of 10 μm as move unit, and the results show that this method effective reduced the small angle between the system optical axis and the measured body in high-precision image measuring system, the evaluation accuracy is less than 0.1°, meeting the requirements in high-precision measurement. The proposed method of detecting verticality based on regional image definition can evaluate the verticality error between the axis of the shooting system and the measured object accurately, effectively and conveniently.