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.