The correlations between water state and the key factors affecting sourdough quality, including cell activity and acidification capacity of lactic acid bacteria (LAB), were established in this study. Results revealed that with the increase of frozen storage time, the cell density (CD), total titratable acidity (TTA), and organic acids content declined, whereas the pH value rose. Further, the freezable water content (FWC) and free water quantity (FWQ) decreased, but the total water loss rate (WLR) and immobilised water quantity (IWQ) increased. The CD showed a highly inversely correlation with WLR, and the pH value was strongly inversely correlated to the FWQ. The confocal laser scanning microscopy (CLSM) observed that the ice crystals had larger volumes during frozen storage. Our data, for the first time, disclosed that the total water content and the FWQ may play a crucial role in maintaining the viability and acidification capacity of LAB in frozen sourdough.