One of the widely used methods for performance analysis of an optical system is the determination of the Modulation Transfer Function (MTF). The MTF represents a quantitative and direct measure of image quality, and, besides being an objective test, it can be used on concatenated optical system. This paper presents the application of software called SMTF (software modulation transfer function), built in C++ and Open CV platforms for MTF calculation on electro-optical system. Through this technique, it is possible to develop specific method to measure the real time performance of a digital fundus camera, an infrared sensor and an ophthalmological surgery microscope. Each optical instrument mentioned has a particular device to measure the MTF response, which is being developed. Then the MTF information assists the analysis of the optical system alignment, and also defines its resolution limit by the MTF graphic. The result obtained from the implemented software is compared with the theoretical MTF curve from the analyzed systems.