A bicycle trainer fastening device, disposed on a bicycle trainer, includes a hollow tube, a first fastening member and a second fastening member. The hollow tube includes a first assembly portion and a second assembly portion at two opposite ends thereof. The first fastening member is disposed correspondingly to the first assembly portion, and includes a first through opening correspondingly in communication with the hollow tube and having an aperture corresponding to an inserting shaft of a bicycle. The second fastening member is disposed correspondingly to the second assembly portion, and includes a second through opening correspondingly in communication with the hollow tube and an aperture corresponding to the inserting shaft. By selecting the first through opening and the second through opening corresponding to the shaft diameter of the inserting shaft and replacing the first fastening member and the second fastening member, the scope of applicability can be maximized.