Most frame structures in mechanical equipment are connected together by bolts, and the occurrence of non-uniform preload between bolts will have a significant impact on the vibration characteristics of frame structures (such as frequency response function (FRF)). In this paper, a FRF calculation method for frame structures considering non-uniform bolt preload is described based on finite element method. Firstly, according to the definition of FRF, the calculation formula suitable for FRF of bolted frame structure is derived based on the principle of modal superposition. Then, based on ANSYS platform, the simulation of bolt non-uniform preload and the method of extracting the stiffness and mass matrices of frame structure containing non-uniform preload are described in order to serve the demand of FRF calculation. Furthermore, the calculation procedure of FRF of bolted frame structure based on ANSYS and Matlab is presented. Finally, a case study of a frame structure with four connecting bolts is carried out to prove the rationality of the proposed FRF calculation method. On this basis, the influence of bolt preload on FRF of frame structure is analyzed by experiment and simulation. The results show that the natural frequency and peak values of FRF increase with the increase of bolt preload.