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Temperature and humidity (TH) are highly important factors that can control the secondary structure and characterization of silk fibroin (SF) biomaterials. In this study, the water stability, secondary structure, mechanical properties, surface morphology, and degradation of silk fibroin films (SFFs) with post‐processing in different TH were investigated. Fourier transform infrared indicated that the SFF secondary structure did not change under low‐relative humidity (RH) despite temperatures up to 180°C, while it transformed at 40°C with 100% RH in 10 min. A film with a higher tensile strength (42.1 ± 8.2 MPa) could be obtained after post‐processing at 90°C/100% RH for 10 min. While a film with higher ductility (elongation at break: 198.8 ± 31.8%) was generated after post‐processing at 40°C/100% RH for 10 min. Scanning electron microscope showed that the film presented a network structure of nanoparticles in series under certain TH post‐treatment. Enzymatic hydrolysis proved that the SFFs containing a higher content of silk II structure degraded more slowly. Therefore, TH post‐treatment is a relatively mild way to change the secondary structure and properties of SFFs, which can be widely used in loading drugs and maintaining the activity of drugs in SF biomaterials. [ABSTRACT FROM AUTHOR]