Objective:To determine neutralizing antibodies against Japanese encephalitis virus (JEV) among healthy people of Southeast Gansu province, and to evaluate immunologic barrier, and provide the data for Japanese encephalitis (JE) control.Methods:Healthy people were divided into the following 3 groups: less than 14 years, 15-39 years, and above 40 years in 2018 in four cities (Pingliang city, Qingyang city, Longnan city, Tianshui city) of Southeast Gansu province. Serum samples of the participants were obtained, and JEV antibody was detected by plaque reduction neutralization assays. The JEV neutralizing antibody positive rates among different groups were compared by chi square test.Results:A total of 1 590 people were investigated. The total positive rate of JEV neutralizing antibody was 28.81%, and the geometric mean titer (GMT) of JEV neutralizing antibody was 1∶27.09. The antibody positive rates of 0-14, 15-39 and ≥ 40 years groups were 32.96%, 20.32%, 30.63%, the difference of antibody positive rates among different age groups was statistically significant ( χ2=22.29, P<0.001). The antibody positive rates in Pingliang city, Qingyang city, Longnan city, Tianshui city were 29.62%, 22.22%, 33.00%, 30.30%, the difference of antibody positive rates among cities was statistically significant( χ2=12.39, P=0.006). Conclusions:The positive rates of neutralizing antibodies against JEV was low among healthy people in Southeast Gansu province, and there is an epidemic risk of JE.