Abstract: A method for testing focal spot of a microfocus X-ray machine was put forward, wherein a knife edge tool was used. The magnified image of knife edge tool was acquired and the unsharpness curve was extracted. The unsharpness value was determined by criteria proposed by Klasens and Morgan, and the corresponding focal spot was measured. In addition, the testing results were verified by duplex wire image quality indicator (IQI) and a wedge shaped resolution card. The results show that the relationship between focus size of the microfocus X-ray machine and applied tube current can be described with certain quadratic formulas approximately in the case of a constant tube voltage. Besides, the result from knife edge tool accords with indications from the duplex wire IQI and wedge shaped resolution card. The measuring method by knife edge tool is able to provide reliable result, nevertheless, the criterion to quantitate the corresponding unsharpness curve should be chosen appropriately. Concerning the criterion, Morgan’s method is more suitable and reasonable.