To research the adhesive properties of polyurethane sealant under shear fatigue loading, a test method for evaluating the adhesive properties of sealants was developed based on the in-situ working condition. Direct tension tests at low temperature were conducted on the samples subjected to various shear fatigue cycles, the effects of various sealants on the maximum tensile force, failure displacement and force-displacement curves were analyzed and compared between experienced and un-experienced shear fatigue loading. The results show that the isocyanate index R of the samples un-experienced shear fatigue loading presents the positive correlation with bonding strength, and negative correlation with deformation resistivity. However, the R value presents a negative correlation with fatigue resistivity of sealants for the samples experienced shear fatigue loading. The adhesive properties and discreteness of sealants were improved due to chain orientation within sealants at the initial period of fatigue cycles. During the fatigue cycles, the stability of maximum force index is superior to the failure displacement due to the discontinuous medium of sealant body. As the increase of fatigue cycles, the accumulation of damage breaks the integrality of the bonding interface, which leads to the change of failure mode from brittle failure to ductile failure. Based on the test results, the sealants with R values between 1.7~2.5 could present better shear fatigue resistance performances.