The combine harvester with multi-cylinder has a wide application in improving the operation efficiency. Therefore, as an important process in combine harvester, threshing operated by multi-cylinder usually makes the working structures suffer hit from the blockage of stones or materials. Consequently, the cylinders fail to guarantee the original balance. Several studies have been performed in this area, based on the dynamic balance method and vibration signal processing. However, most of them are not efficient for multi-cylinder. This study aims at developing an online dynamic balance detection system for multi-cylinder in combine harvester, in order to enhance the efficiency and accuracy of online dynamic balance detection. A multi-cylinder test rig is developed, and unbalanced vibration experiments under different working conditions are performed. The results show that the different measuring positions have different sensitivities to unbalanced mass. The vibrations will be transmitted through the frame, and coupled with each other. Under the multi-cylinder rotation, the vibration has strong randomness and poor convergence. For this purpose, the single measuring point overall balancing method is proposed. An adaptive multiple filter algorithm relying on the LabVIEW platform, is also proposed. Through phase relation between cylinders, the single measuring point overall balancing algorithm is developed. The comparison experimental results show that the proposed algorithm is more robust under the multi-cylinder rotation. The proposed method simultaneously installs sensors on the bearing seat of No. 1 tangential cylinder and balance 3 cylinders. The traditional method balance 3 cylinders separately change the sensor position. The effect of the proposed method is almost 76%, while that of the traditional balance method is almost 66%. For the multi-cylinder system, the single measuring point overall balancing method can efficiently improve the balance effect by 10%. Finally, it can be deduced from the experiment on the combine harvester that the accuracy of the proposed method reaches G3.9.