The slug test has been commonly used to estimate aquifer parameters. Previous studies on the slug test mainly focused on a single-layer aquifer. However, understanding the interaction between layers is particularly important when assessing aquifer parameters under certain circumstances. In this study, a new semi-analytical model on transient flow in a three-layered aquifer system with a partially penetrating well was developed for the slug test. The proposed model was solved using the Laplace transform method and the Goldstein-Weber transform method, where the semi-analytical solution for the model was obtained. The drawdowns of the proposed model were analyzed to understand the impacts of the different parameters on the drawdowns in a three-layered aquifer system. The results indicated that groundwater interactions between the layers have a significant impact on the slug test. In addition, a shorter and deeper well screen as well as a greater permeability ratio between the layers creates a greater interface flow between them, leading to a higher drawdown in the slug test. Finally, a slug test in a three-layered aquifer system was conducted in our laboratory to validate the new model, which indicated that the proposed model performed better in the interpretation of the experimental data than a previous model proposed by Hyder et al. (1994). We also proposed an empirical relationship to qualitatively identify the errors in the application of single-layer model for the analysis of response data in a three-layered aquifer system.