Purpose: To verify the feasibility of using anterior segment optical coherence tomography (AS-OCT) to measure the lower lid margin thickness (LLMT) from posterior lash line to the mucocutaneous junction. Methods: This was a prospective, single-center, observational study. Fifty-six volunteers between 20 and 79 years old with normal healthy eyes were included in this study. Using AS-OCT to measure the LLMT at the middle position was separately performed by two operators under the same conditions. Results: The mean age of 56 subjects (24 males and 32 females) was 45.4 ± 15.7 years. The LLMT measured by AS-OCT between two operators was 0.86 ± 0.23 mm and 0.85 ± 0.22 mm, respectively. The reliability and repeatability between two operators were evaluated by intraclass correlation coefficient (ICC = 0.97). Based on one operator’s measurements, the LLMT was 0.70 ± 0.10 mm at ages 20–29, 0.68 ± 0.09 mm at ages 30–39, 0.94 ± 0.12 mm at ages 40–49, 0.93 ± 0.20 mm at ages 50–59, 1.03 ± 0.15 mm at ages 60–69, and 1.24 ± 0.19 mm at ages 70–79, which indicated a significant positive correlation with age (rs = 0.74, p < 0.001). In addition, there was a significant difference in the LLMT between genders (0.94 ± 0.26 vs. 0.80 ± 0.19 mm, p = 0.03). Conclusions: A good agreement between two operators indicated that it seemed to be feasible to use AS-OCT to measure the LLMT. Additionally, we found that the LLMT appeared to be closely related to age and gender.