In order to study the long-term loss law of anchorage force and the calculation method of prestress loss of anchorage force, a calculation method of the prestress loss of anchorage force was proposed to account for three factors. The proposed method is based on the generalized Hooke’s law, relaxation rate time-history response equation and improved coupling constitutive model. It was adopted to calculate the theoretical loss value of the anchor cable prestress coupled with the internal sliding lock of the anchor plate, the stress relaxation of the cable body steel strand, and the rock mass creep, respectively. In order to verify the accuracy of the long-term loss calculation method, the anchor cable pull-out failure test was also carried out. By analyzing the transfer mechanism of the pull-out load, it was determined that the pull-out stage of the free section of the load-incremental displacement curve was the working load interval of the anchor cable. The median value of the working load interval was taken as the effectiveness force of the anchor cable, and the measured value of the long-term loss of prestress was also obtained. Results show that the prestress loss of the anchor cable is about 17.5%-27.5% during 20 years. The rock mass creep is the most significant factor affecting the long-term loss of the anchor cable. Besides, there is an error of about 4.2% between the calculated value of the three-factor loss calculation method and the measured value of prestress loss at site. This verifies the accuracy of the three-factor loss calculation method, and also shows that the three-factor calculation method of long-term prestress loss mentioned here meets accuracy requirements and is feasible.