An anti-loose performance test method in fastener thread glue coating. A fastener coated with an anaerobic adhesive is used for connection according to an actual installation state, and is compared with a test piece not coated with the anaerobic adhesive. The fasteners of different states are fixed on a high or low temperature tool, so as to simulate a real thermal environment (including a normal temperature, a low temperature and a high temperature) of the fastener. The whole set of the tool is fixed on a test bench and carries out a vibration test according to a vibration condition in a corresponding work environment, so as to simulate a real mechanical environment of the fastener. In order to test the anti-loose performance, a torque check is carried out before the test, and the torque is re-checked after the vibration test is finished. The method of the invention can effectively test the anti-loose performance of the fastener in the high temperature, low temperature, and vibration environment after the fastener is coated with the glue.