Log is widely used in Chinese traditional wooden buildings, which has good compression, bending, tensile and seismic performance, but the existence of knots in log will have a greater adverse impact on the structure. Taking the residence of Zhou Fujiu, a salt merchant in Yangzhou as an example, the building was built in the late Qing Dynasty, covering an area of 　3700 square meters. Through the field investigation of Zhou Fujiu building in Yangzhou, it is found that in addition to the common natural cracks, the wooden beams with knots in the tension zone are obviously damaged. In order to further study the influence of knots on the seismic performance of wooden beams and provide relevant theoretical basis for the protection of traditional Chinese wooden buildings, six different wooden beams were designed according to the scale of 1:5.28, and the low cycle reciprocating loading test under three-point loading was carried out. The effects of knots at different positions and depths on the seismic performance of timber beams are simulated respectively. The hysteretic curve and skeleton curve of the timber beam with knots are obtained by experiments, and the stiffness degradation and energy dissipation capacity are analyzed. The results show that with the increase of the depth of the knots, the bearing capacity of the beam shows a significant decreasing trend, the hysteretic curve becomes more stable, the energy dissipation capacity decreases, and the seismic performance decreases. When there are knots on both sides of the bending and compression area, the seismic performance of the wooden beam decreases most significantly.