We investigate the influence of the optical fiber fabrication process on the fluorescence lifetime of Er³⁺ and Tm³⁺ ions. Optical fiber preforms were prepared using the MCVD method combined with Al₂O₃ nanoparticle doping. The preforms were subjected to various fabrication processes, such as preform elongation, fiber drawing, and heat treatment. The matrix structure of the preforms and fibers was studied by XRD and TEM. The fluorescence lifetime was measured. The fabrication processes caused the dissolution of the doped Al₂O₃ nanoparticles and a significant decrease of fluorescence lifetime of Tm³⁺ ions, from 875 µs in the preform down to 610 µs in the fiber, whereas no significant effect was observed for Er³⁺ ions, with fluorescence lifetime in the 9.6–10.2 ms range.