Objective To analyze the vitamin D levels in alopecia patients of different genders and the correlation with clinical characteristics. Methods A total of 50 alopecia patients who were treated in our hospital from December 2022 to August 2024 were selected, including 25 males and 25 females. The levels of vitamin D and testosterone in alopecia patients of different genders were analyzed, and the relationship between testosterone and vitamin D was evaluated by Pearson correlation analysis. In addition, the relationships between vitamin D and relevant clinical characteristics, as well as antinuclear antibodies in alopecia patients of different genders were analyzed. Results The levels of vitamin D and testosterone in female patients were lower than those in male patients (P <0.05). There was a negative correlation between testosterone and vitamin D in both male and female alopecia patients (P <0.05). However, there was no correlation between vitamin D and relevant clinical characteristics, as well as antinuclear antibodies in male and female alopecia patients (P >0.05). Conclusion Vitamin D deficiency may be one of the pathogenesis of alopecia. Testosterone is closely related to vitamin D, but there is no correlation between vitamin D and relevant clinical characteristics, as well as antinuclear antibodies in alopecia patients of different genders.