The study aims to assess the diagnostic and clinical significance of autoantibodies against aquaporin-1 (anti-AQP1) and aquaporin-5 (anti-AQP5) in primary Sjögren's syndrome (pSS). A total of 163 participants were categorized into three groups: pSS group, other connective tissue diseases (CTD) group, and healthy control (HC) group. The levels of anti-AQP1 and anti-AQP5 autoantibodies in serum were determined using enzyme-linked immunosorbent assay (ELISA), and clinical data from patients were collected for statistical analysis. Our results showed that the level of anti-AQP1 in the pSS group was higher than in the HC group (P < 0.05), and no significant difference was observed between the pSS group and the CTD group (P > 0.05). ROC showed that the anti-AQP1 had no diagnostic value for pSS (P > 0.05). The anti-AQP5 level of 39 healthy adults was all below the cut-off value (14.10ng/ml) (P < 0.05). The level of anti-AQP5 in the pSS group was higher than the CTD group (P < 0.05), the AUC was 0.86 (95% CI 0.80-0.93), with a sensitivity of 0.95 (95% CI 0.87-0.99) and a specificity of 0.70 (95% CI 0.58-0.84). No correlation was found between anti-AQP5 levels and the EULAR primary Sjögren's syndrome disease activity index score, anti-SSA, anti-SSB, antinuclear antibodies, rheumatoid factor, anti-ds-DNA, salivary gland flow rate, complement 3, and lymphocyte count in pSS samples (P > 0.05), respectively. Therefore, the elevated anti-AQP5 may emerge as a novel diagnostic biomarker for pSS patients due to high sensitivity and specificity.