The purpose of this study was to explore the feasibility of using optical coherence tomography (OCT) for real-time and quantitative monitoring of enamel development in gene-edited enamel defect mice. NF-κB activator 1, known as Act1, is associated with many inflammatory diseases. The antisense oligonucleotide of Act1 was inserted after the CD68 gene promoter, which would cover the start region of the Act1 gene and inhibit its transcription. Anti-Act1 mice, gene-edited mice, were successfully constructed and demonstrated amelogenesis imperfecta by scanning electron microscope (SEM) and energy dispersive X-ray (EDX) spectroscopy. Wild-type (WT) mice were used as the control group in this study. WT mice and anti-Act1 mice at 3 weeks old were examined by OCT every week and killed at eight weeks old. Their mandibular bones were dissected and examined by OCT, micro-computed tomography (micro-CT), and SEM. OCT images showed that the outer layer of enamel of anti-Act1 mice was obviously thinner than that of WT mice but no difference in total thickness. When assessing enamel thickness, there was a significant normal linear correlation between these methods. OCT could scan the imperfect developed enamel noninvasively and quickly, providing images of the enamel layers of mouse incisors.