Understanding the microstructure change of polymer nanocomposites (PNCs) under elongation deformation at the molecular level is the key to coupling structure-property relationships of PNCs. In this study, we developed our recently proposed in situ extensional rheology NMR device, Rheo-spin NMR, which can simultaneously obtain both the macroscopic stress-strain curves and the microscopic molecular information with the total sample weight of ∼6 mg. This enables us to conduct a detailed investigation of the evolution of the interfacial layer and polymer matrix in nonlinear elongational strain softening behaviors. A quantitative method is established for in situ analysis of (1) the fraction of the interfacial layer and (2) the network strand orientation distribution of the polymer matrix based on the molecular stress function model under active deformation. The results show that for the current highly filled silicone nanocomposite system, the influence of the interfacial layer fraction on mechanical property change during small amplitude deformation is quite minor, while the main role is reflected in rubber network strand reorientation. The Rheo-spin NMR device and the established analysis method are expected to facilitate the understanding of the reinforcement mechanism of PNC, which can be further applied to understand the deformation mechanism of other systems, i.e., glassy and semicrystalline polymers and the vascular tissues.