One of the persistent challenges in applying hydrogels in the biomedical fields is strengthening their stability and rheological properties, particularly their modulus, since natural tissues exhibit much greater toughness than hydrogels derived from biomaterials. Modulus enhancement can be achieved by optimizing the chemical cross-linking conditions such as treatment temperature during hydrogel synthesis. This study investigates the influence of temperature on the properties of chitosan and oxidized pullulan (CS/APUL) hydrogels via chemical crosslinking for biomedical applications. Fourier transform infrared spectroscopy, thermal analysis, scanning electron microscopy, and rheological analysis were used to examine the physicochemical properties of CS/APUL hydrogels. The results demonstrate that temperature significantly affects the onset gelation time, gel fraction, and gel strength of CS/APUL hydrogels. As the temperature increased from 25 to 45 °C, the gelation time decreased from 117.25 ± 4.40 to 21.3 ± 1.10 s, indicating faster crosslinking and network formation. Concurrently, the gel fraction and complex modulus (gel’s strength) of the hydrogels increased from 56 to 68%, and 893.57 ± 33.7 to 1779.75 ± 72.7 Pa, respectively, suggesting enhanced mechanical stability and structural integrity at higher temperatures. Conversely, the swelling ratio of the hydrogels decreased with rising temperature, likely due to the formation of a denser and more tightly crosslinked network. These findings highlight the tunability of CS/APUL hydrogels through temperature control, making the biomaterials promising candidates for applications such as drug delivery, tissue engineering, and wound healing, where precise mechanical and swelling properties are crucial. (Figure presented.)