Bamboo fibers (BFs) at different ages (1, 3, and 5 years old) were prepared by pretreatment and steam explosion. The results demonstrated that the separation degrees of BFs increased with the age of the bamboo. The combined effects of pretreatment and steam explosion partially removed the hemicellulose and lignin from BFs, while the crystalline structure of cellulose remained unaltered. The water vapor adsorption isotherms of BFs at different ages all showed a typical S-shape and could be classified as type II, and the hysteresis of BFs increased with the age of the bamboo. After pretreatment and steam explosion, the tissues in the vascular bundle were effectively crushed, and the parenchyma tissues were dislodged and separated from the fiber sheath. During the steam explosion process, hot steam penetrated the bamboo’s pores, including the pores induced by pretreatment. This caused fractures and delamination in the interface between the vascular bundle and the parenchyma tissues. These cracks progressed layer by layer along the vascular bundle interface, leading to the fracture of the bamboo.