〔Abstract〕 Objective To establish the reference interval of serum 25-hydroxy vitamin D [25 (OH) D] in apparent healthy people in Fuzhou area, and analyze the difference of serum 25 (OH) D between different genders and ages. Methods A total of 401 healthy people who underwent physical examination in Union Hospital Affiliated to Fujian Medical University from January 2016 to January 2021 were selected. The level of serum 25 (OH) D was detected by electrochemiluminescence method. Statistical analysis was performed to establish reference intervals. Results The reference interval of serum 25 (OH) D established in this study was 12.30-44.87 ng·mL-1. Grouped by gender, the serum 25 (OH) D concentration of males was higher than that of females, the difference was statistically significant (P < 0.05). Grouped by age, there was no significant difference in serum 25(OH)D concentration between 30-39 years old and 40-49 years old group (P > 0.05), while the difference between the age among other groups was statistically significant (P < 0.05). Conclusion The reference interval of serum 25 (OH) D for healthy people in Fuzhou area established in this study was 12.30-44.87 ng·mL-1. The level of serum 25 (OH) D was related to gender and age. The level of serum 25 (OH) D was higher in men than in women and increased with age.