Objective To analyze the factors influencing serum 1,5-anhydro-D-glucitol (1,5-AG) levels and establish sex-specific reference intervals for 1,5-AG. Methods A total of 873 participants, aged 20 to 72 years, were enrolled from an epidemiological survey conducted between October and December 2020 in Nanjing, Jiangsu Province, and surrounding areas. General clinical data were collected via questionnaire. Physical examinations and laboratory assessments—including serum 1,5-AG and other routine clinical biochemical indicators—were conducted by trained professionals. Participants were categorized into different disease groups according to inclusion and exclusion criteria. After log-transformation of non-normally distributed data, Student's t-test was used to compare serum 1,5-AG levels among these groups. Pearson correlation analysis and multivariate linear regression were employed to assess associations between serum 1,5-AG and various clinical parameters. The reference interval for 1,5-AG was determined based on the 5th percentile among the apparently healthy population. Results Serum 1,5-AG levels were significantly associated with sex, body mass index (BMI), body roundness index (BRI), fasting blood glucose, fasting insulin, 2-hour postprandial blood glucose, glycated hemoglobin (HbA1c), estimated glomerular filtration rate (eGFR), total cholesterol, and carbohydrate-derived energy intake (all p < 0.05). Participants were divided into nine groups: apparently healthy, diabetes mellitus, hypertension, hyperlipidemia, impaired fasting glucose (IFG), impaired glucose tolerance (IGT), hepatic dysfunction, renal dysfunction, and obesity. Significant differences in 1,5-AG levels were observed between the apparently healthy group and the hyperlipidemia, hepatic dysfunction, and obesity groups. The sex-specific reference intervals for serum 1,5-AG, defined by the 5th percentile in the apparently healthy population, were >99.8 μmol/L for males and >62.9 μmol/L for females. Conclusion Serum 1,5-AG levels are associated with glycemic status, lipid metabolism, and dietary patterns, and are influenced by age, sex, renal function, and dietary composition. Males exhibited significantly higher serum 1,5-AG levels than females; however, no significant differences were found across different age groups. The sex-specific reference intervals for serum 1,5-AG established in this study provide a valuable tool for monitoring glycemic variation and guiding the clinical management of diabetes.