X-ray Nano-Computed Tomography (Nano-CT) is widely used in micro device nondestructive testing, material science, life science and other applications fields. Micro-focus X-ray source is one of the key components of Nano-CT and is directly affected by the quality of electron beam. Diameter is the key performance index of electron beam. The detection of electron beam spot diameter is of great significance for monitoring the performance of Nano-CT and quantitatively evaluating the qualities of design and fabrication of the electron gun and electron optical system. In this paper, the diameter direct detecting method is presented and applied to the FEI Quanta 600 SEM. The relationship between electron beam diameter and detecting curve is analyzed at first, then the application feature of diameter detecting methods, including crosshair detecting method, slit detecting method and single edge detecting method, is researched and compared. Furthermore, the experimental results demonstrate that the edge detection has higher detecting accuracy and lower requirement on the sampler, and that after computation the detecting error is within 20 nm.