Alpha‐fodrin, an intracellular organ‐specific cytoskeleton protein, was identified recently as an autoantigen associated with Sicca‐ and Sjögren's syndrome (SS). Identification of the antigenic determinants of α‐fodrin is a prerequisite to developing highly sensitive and specific anti‐α‐fodrin antibodies, which provides potential means for the diagnosis of primary Sjögren's syndrome (pSS) in patients. Based on the structure and predicted antigenic sites of α‐fodrin protein with 560 amino acids (α‐fodrin 560), we prepared a set of overlapping recombinant protein fragments covering antigenic epitopes and synthesized a set of peptides derived from the α‐fodrin protein. These recombinant proteins and synthesized peptides were subjected to screening with pSS patients sera, respectively. The peptide with the strongest immunoreactivity was used as antigenic peptide to define further the role of anti‐α‐fodrin‐peptide antibodies in the sera of 135 patients with pSS, 48 patients with systemic lupus erythematosus (SLE), 88 patients with rheumatoid arthritis (RA) and 83 normal controls. Our data showed that the N‐terminal peptide of amino acids 46–59 (N46) of α‐fodrin 560 was the epitope with strongest antigenicity. The prevalences of anti‐N46 peptide antibodies (α‐N46PA) in patients with pSS, SLE, RA and normal controls were 78·5%, 10·4%, 21·6% and 6·0%, respectively. The sensitivity and specificity of the autoantibodies in pSS were 78·5% and 86·8%, respectively. These results suggest the α‐N46PA which shows highest sensitivity and specificity is of significance to develop an effective diagnostic approach for pSS.