Several Ce3+/Tm3+-co-doped alumino-silicate glasses with various Ce3+/Tm3+ ratios were prepared by a sol-gel method combined with vacuum sintering. To determine their radiation resistance, the glasses were exposed to 1 and 100 kGy γ-ray. The effect of the Ce3+/Tm3+ ratio on the absorption, emission and fluorescence lifetime of the glasses under investigation, was examined before and after irradiation. The Ce4+ and Ce3+ cations were detected and the nature of Si-E′ and AlOHC color centers caused by irradiation was identified by radiation-induced absorption (RIA) and electron paramagnetic resonance (EPR) spectroscopy. Co-doping with Ce3+ ions can significantly improve the radiation resistance of the glass, although it can deteriorate the spectral properties of Tm3+ ions. Additionally, the related influence mechanisms are discussed. The obtained results indicate that Ce3+/Tm3+-co-doped alumino-silicate glasses with optimized Ce3+ ion concentrations, a Ce3+/Tm3+ molar ratio less than 0.3, can be used as an active medium for radiation-resistant materials in harsh radiation environments.