The increase in railway coverage significantly increased the railway mileage in karst areas. However, the fragile ecosystem in karst areas and weak mechanical performance in cavities cause hidden railway safety hazards. Even with the crucial stability and safety of railways in karst areas, there is only limited research conducted on the dynamic response of railway subgrade and the influence of hidden soil holes on the stability under different conditions. In this study, the Guizhou section of the Shanghai–Kunming high-speed railway was chosen as the research area. On-site monitoring and simulations using ABAQUS were performed to study the dynamic response of the subgrade under the dynamic load of a train. The influence of the soil hole, axle load, and vehicle speed on the dynamic response law and stability of the subgrade were considered based on the model rationality verification. Previous studies have demonstrated the considerable impact of soil holes on the dynamic response characteristics and stability of roadbeds. The ambient vibration frequency of the train was concentrated at 10–60 Hz. Under train load, the amplitude of the dynamic response parameters of each subgrade structural layer increased with the axle load; however, the increase in speed exhibited a greater impact on the vertical dynamic displacement and dynamic stress than on the dynamic acceleration, which affected the stability of the soil hole and roadbed. This study summarized the dynamic response characteristics of subgrade in karst areas under different conditions, thereby providing a design reference for high-speed railways in karst areas.