Abstract Background The incidence of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and primary biliary cirrhosis has increased significantly in China. Information about the susceptibility or potential of autoimmune diseases in the general population is lacking. Objective To explore the prevalence of antinuclear antibody (ANA) and its specificities in the general population in China. Methods Twenty thousand nine hundred seventy sera samples were taken from the physical examination center in Baoding, China. Indirect immunofluorescence and line immunoassays were used to detect ANA and its specificities, respectively. Results Samples from females had a higher prevalence of ANA than samples from males (χ2 = 278.55; P < 0.01). For both sexes, the prevalence of ANA positively correlated with age and there were significant differences among different age groups at 10-year intervals, except the 80 years group ( P < 0.05). One thousand two hundred forty-three ANA-positive samples were further analyzed with line immunoassays. There was a significant difference among age groups and between sex groups in terms of the specific autoantibodies ( P < 0.01). The autoantibodies with the top-3 positive frequencies were anti-Ro-52, anti-M2, and anti-SSA. Conclusions There was a high prevalence of ANA positivity in the general Chinese population that seemed to be influenced by sex and age and correlated with specific autoantibodies.