The adeno-associated virus 9 (AAV9) vector was particularly notable for its broad tissue tropism, making it a preferred vector for gene therapy. Goals: The study aimed to investigate the patterns of pre-existing immunity against AAV9 in the Chinese population. In this study, we conducted a serological research from November 2022 to June 2024. The study included 341 participants in total with age ranged from 0 to 90 years old: 270 healthy individuals, 30 pediatric patients and 41 adults with rare diseases. Total AAV9-binding antibodies (TAbs) and neutralizing antibodies (NAbs) were measured. The seroprevalence of anti-AAV9 NAbs showed no significant differences between healthy individuals and rare disease patients across both pediatric and adult groups. Newborns exhibited a high NAb-positive rate (64.3 %), while children aged 6 months to 3 years had the lowest prevalence (7.7 %). This rate progressively increased through childhood and adolescence. Overall, 58.7 % of the Chinese population aged 0-90 years tested positive for anti-AAV9 NAbs, with adults showing a significantly higher prevalence than children (75.0 % vs. 34.3 %). Additionally, 58.1 % of the population exhibited low levels of anti-AAV9 NAb titers (IC