Abstract: The serum bactericidal assay (SBA), a gold standard recommended by the World Health Organization for detecting antibody levels against Neisseria meningitidis (Nm), was utilized in this study. Based on the classic Nm bactericidal antibody detection methodology, we optimized experimental parameters including sample volume, complement selection, Nm target bacterial concentration, bactericidal reaction time, and positive reference serum concentration to establish a serogroup X Nm bactericidal antibody detection protocol. The specificity, repeatability, and stability of the positive reference serum were systematically validated. The established method was applied to analyze bactericidal antibody levels in 508 serum samples of healthy individuals under 5 years old from Hennan, Jiangsu, Anhui and Shanxi provinces. Geometric mean titers (GMT) were calculated and statistically analyzed by region and age group. Results demonstrated that 95.7% serum samples exhibited serogroup X Nm bactericidal antibody titers below 8, with an overall GMT of 2.20. No statistically significant regional differences in GMT were observed (H = 6.688, P = 0.083). Age-stratified analysis revealed that 6.37% of infants aged 0-11 months displayed antibody titers greater than or equal to 8. This proportion progressively declined with age, reaching 0.49% in the 2-5 years group. However, no significant inter-group differences in GMT were detected among age cohorts (H = 4.756, P = 0.093). The findings indicate that the serum bactericidal antibody titers against Neisseria meningitidis serogroup X (Nm X) are relatively low among healthy children under 5 years of age in China. This study has developed a reliable detection method for Nm X SBA with good specificity, stability, and reproducibility. Furthermore, it has provided the data on Nm X SBA levels among children under 5 years old from various regions, offering scientific guidance for the future prevention and control of serogroup X meningococcal disease in China. All the serum used in this experiment were approved by Henan Provincial Center for Disease Control and Prevention Ethics Review Committee (approval number: 2019YM-005-02), Jiangsu Provincial Center for Disease Control and Prevention Ethics Review Committee (approval number: JSJK202-A054-02), and Shanxi Provincial Center for Disease Control and Prevention Ethics Review Committee (approval number: SXCDCIRBP2020050001).