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This study investigates the sound insulation performance of an anechoic chamber, exploring the influence patterns of different multilayer material combinations on wall sound insulation characteristics. Based on sound transmission theory, a predictive model for multilayer material wall sound insulation was established. The finite element method was employed to simulate the sound propagation characteristics of walls and glass doors with various material combinations. After validating the simulation results through a double-room method experiment, the material combination scheme for the anechoic chamber walls and glass doors was optimized. Based on this, a 1000 mm × 1000 mm × 2300 mm soundproof room prototype was designed and constructed. Its sound insulation performance under reverberant conditions was tested using the insertion loss method and compared with simulation data. Simultaneously, a hybrid calculation method combining low-frequency finite element analysis with high-frequency statistical energy analysis enabled precise and efficient prediction of the overall sound insulation performance of the soundproof room. Research revealed that single-pane glass with thicknesses between 5 and 20 mm conformed to the mass law, with sound insulation increasing by an average of 0.8 dB per additional millimeter. The 10 mm single-pane glass emerged as the optimal choice for the soundproof room’s glass door due to its ideal thickness and excellent low-to-mid-frequency sound insulation. The optimized wall structure featured compact thickness, outstanding low-frequency sound insulation, and balanced mid-to-high-frequency performance. Simulation and experimental results for the core frequency range of 63–1000 Hz showed high consistency, which validates the reliability of the theoretical model and simulation methodology within this frequency band. The deviation of simulation results from experimental data in the frequency range above 1000 Hz is mainly caused by acoustic leakage due to experimental sealing defects, and the high-frequency simulation results are only used for trend analysis rather than conclusion support. This study identifies the optimal multi-layer material combination for soundproof rooms, providing practical material strategies for acoustic design. It also reveals the sound insulation mechanisms of multi-layer composite structures. The findings offer significant reference for optimizing soundproofing materials and structures in architectural acoustics and transportation noise control.