The 2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO) oxidized cellulose film (TOCF) has been attempted to be used as a substrate in electronic and optoelectronic devices, but the changes in the TOCF properties before and after annealing treatment have usually been neglected during device fabrication. In this study, TOCF was treated in different atmospheres (air, vacuum, and N2) and at different temperatures, and the properties were investigated. The results indicate that the optical properties are slightly affected by atmosphere and temperature; only slight transmittance loss and haze increase have been observed when TOCF is exposed to an air atmosphere at temperatures of above 120℃. In contrast to the slight effects on the optical properties, cellulose degradation and a loss of film strength have been observed regardless of the atmosphere used when placed at temperatures of above 100℃. Specifically, TOCF was exposed to air, followed by N2 and vacuum atmospheres. Additional Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) results showed that increasing the temperature had no significant effect on the structure of TOCF. Therefore, the annealing temperature should be controlled at a temperature of lower than 100℃and a vacuum atmosphere is preferred.