The invention discloses a method for evaluating and detecting the looseness prevention performance of fasteners of transmission towers. The method includes steps of firstly, verifying product parameter characteristics of fastener connection modes; secondly, testing the looseness prevention performance of bolts applied to different places; and thirdly, testing an installation condition to realize the optimal looseness prevention performance. The product parameter characteristics include models and the mechanical strength of the bolts, and a value assigned for each common galvanized bolt is 0.5-0.7 times of load assuredly. The method has the advantages that testing conditions with vibration load consistent with or similar to vibration load on fastening connectors at different positions of towers in different regions can be set according to fastener connection application places and actual installation conditions of the towers, so that the looseness prevention performance of the fastening connectors can be evaluated and tested, optimal model selection for the fasteners of the towers is realized, and effectiveness and applicability of gallop prevention transformation for the towers are improved.