In order to study the dynamic interaction characteristics and rules of pile-soil-nuclear island structure in layered soil, a 3 × 3 pile-foundation model shaking table test was carried out under the distribution of two soil layers: lower hard soil-upper soft soil and lower hard soil-middle soft soil-upper hard soil. This paper mainly discusses the variation of micro-strain of pile with acceleration amplitude under different working conditions in CHICHI seismic records with different amplitudes adjusted. In order to fully study the dynamic interaction of pile-soil-nuclear island structure, the maximum input acceleration amplitude is 0.15 g when the top structure isn’t installed, and 0.5 g when the top structure is installed. The test results show that pile-soil is mainly in a state of motion interaction when there is no upper nuclear island structure. In this case, the maximum micro-strain of pile in two-layer soil is located at the top of pile, and the peak micro-strain of pile in three-layer soil is located at the upper interface of soil layer. When the maximum input value is 0.15 g, the pile in both soil layers is in the elastic stage. When there is a superstructure, the pile-soil-superstructure is mainly in a state of inertial interaction, and the micro-strain of the pile increases significantly. In the two-layer soil test condition, the concrete at the top of the pile begins to crack when the input ground motion amplitude is 0.2 g; in the three-layer soil test condition, the concrete at the top of the pile begins to crack when the input ground motion amplitude is 0.3 g. Under the same amplitude, a layer of hard soil at the top of the pile can obviously reduce the micro-strain and improve the overall seismic performance of the pile.