Hierarchically organized three-dimensional (3D) carbon nanotubes/graphene (CNTs/graphene) hybrid nanostructures hold great promises in composite and battery applications. Understanding the junction strength between CNTs and graphene is crucial for utilizing such special nanostructures. Here, in situ pulling an individual CNT bundle out of graphene is carried out for the first time using a nanomechanical tester developed in-house, and the measured junction strength of CNTs/graphene is 2.23 ± 0.56 GPa. The post transmission electron microscopy (TEM) analysis of remained graphene after peeling off CNT forest confirms that the failure during pull-out test occurs at the CNT-graphene junction. Such a carefully designed study makes it possible to better understand the interfacial interactions between CNTs and graphene in the 3D CNTs/graphene nanostructures. The coupled experimental and computational effort suggests that the junction between the CNTs and the graphene layer is likely to be chemically bonded, or at least consisting of a mixture of chemical bonding and van der Waals interactions.