For understanding the behavior of the active substance in vivo, the near-infrared (NIR) spectral variations of ovalbumin (OVA) loaded in poly(N, N-dimethyl acrylamide) (PDMAA) hydrogel with temperature were investigated. Analyzing the spectra with improved resolution by continuous wavelet transform (CWT), the absorption variation of the peak at 4851 cm-1 arising from the α-helix of OVA with temperature was studied. The results show that a sharp decrease occurs at a lower temperature in PDMAA hydrogel, indicating that the unfolding of OVA in PDMAA hydrogel is facilitated. On the other hand, the intensity changes for the hydrogen-bonded water were consistent with that for the protein, providing evidence for facilitating the unfolding. Furthermore, the spectral feature of a new water structure with two hydrogen bonds was obtained from the spectra of OVA loaded hydrogel by independent component analysis (ICA). By analyzing the difference of the water structure with temperature and the side chain of hydrogels, it is demonstrated that the water structure may be a double hydration water surrounding both the protein and the methyl groups of the hydrogel. The easy dissociation of the double hydration water may be a crucial factor in facilitating the unfolding of proteins within hydrogels.