The special melodious timbre of bamboo musical instruments is closely related to the multi-level, multi-scale, delicate structure of bamboo. In this study, the influence of different excitation surfaces, bamboo height, and bamboo age on acoustic vibration performance was investigated. The mechanism of the influence of the bamboo tissue structure on its acoustic properties was further investigated. The results show that the acoustic conversion efficiency (ACE) of the bamboo-green side is 1307.42 m4/kg·s, which is higher than that of the bamboo-yellow side (1218.60 m4/kg·s). The E'/G' ratio from the bamboo-green side is 8.21, lower than that of the bamboo-yellow side (8.45). The comprehensive acoustic performance near the root (0–1.6 m) of the plant is optimal and has the maximum ACE value; ACE value decreases gradually from root to tip. The comprehensive acoustic properties of 8-year-old bamboo are the best compared to other ages. The ACE values of 2-year-old, 5-year-old, and 8-year-old bamboo are 997.37 m4/kg·s, 1284.19 m4/kg·s, and 1355.82 m4/kg·s with E'/G' of 8.34, 8.64, and 9.49, respectively. A highly oriented vascular fiber is conductive to the vibrational efficiency of bamboo, while loose and porous parenchyma cells play a resonant and tonal role. Highly oriented microfibrils and a large number of pores with diameter > 50 nm create a sound transmission channel and resonance cavity, which is the structural basis of the clear timbre of bamboo. However, a damping dissipation effect is produced affecting the vibration capacity and vibration energy transfer of the cellulose microfibril when abundant pores < 50 nm in size are located in lignin and between hemicellulose and cellulose skeletons. The content of the matrix composed of hemicellulose and lignin in the cell wall creates a chemical material basis that affects the acoustic performance of bamboo. The structural characteristics of bamboo, including directional gradient structure, fiber-parenchyma two-phase composite structure, and muli-scale pore structure, as well as key chemical “rebar-cement-sand” components of the rigid cellulose macromolecular chain, viscoelastic hemicellulose, and lignin have synergistic effects on bamboo acoustic vibration performance.