In this prospective clinical study, the effect of clear aligners on periodontal health and oral hygiene was examined. As the same time, microbial changes of the aligner tray and subgingival microbiota community were investigated. The study recruited fifteen patients, and clinical parameters were recorded at three different time points: before the initiation of aligner treatment (T0), 1month after treatment onset (T1), and 3months after treatment onset (T3). Plaque samples were collected from the inner surface of aligners and subgingival sulcus at each of these time points. The microbial composition of the samples was analyzed using 16S rRNA gene sequencing, and changes were evaluated based on the abundance of amplicon sequence variants (ASVs). Reduction in plaque index and improvement in periodontal health were observed. In aligner tray plaque samples, the relative abundance of Streptococcus increased significantly, as well as the richness and diversity of microbiota decreased substantially as the duration of treatment time. In subgingival plaque samples, alpha and beta diversity of microbiota did not change significantly. During the clear aligner treatment, the patients' periodontium remained in a healthy condition, and clear aligner treatment had no significant impact on the composition of subgingival microbiota. The structure of the aligner tray microbiota altered significantly at both phylum and genus levels and attracted a unique and less diverse microbiota community. Clear aligner treatment has no significant impact on periodontal health and subgingival microbiota composition of patients.