The total RNA of Aquilaria sinensis vitro roots was extracted using the EASYspin Plus Plant Rapid Extraction Kit after three consecutive generations of salt stress treatment, and sequenced using the HiSeq 2500 high- throughput sequencing platform, aiming at exploring the roles of miRNAs in the three generations of A. sinensis vitro roots under salt stress and the metabolic pathways that may be involved in the regulation, and providing references to reveal the mechanisms of A. sinensis. The miRNA expression profiles of the first, second and third generations were obtained by using the high-throughput sequencing platform and bioinformatics technology, and the differential miRNAs in the three generations of vitro roots were screened, and the target genes of the differential miRNAs were predicted, and then the target gene analysis was performed to obtain the miRNAs that might be involved in the secondary metabolites of A. sinensis and those that were related to the response to salt stress. The results showed that 508 known miRNAs and 764 new predicted miRNAs were detected in the first-generation samples; 444 known miRNAs and 673 new predicted miRNAs were detected in the second-generation samples; 904 known miRNAs and 1100 new predicted miRNAs were detected in the third-generation samples. A total of 59 differentially expressed miRNAs and 275 target genes were obtained from the three samples. The up-regulated miRNA target genes were mainly involved in flavonoid biosynthesis and glutathione metabolism, while the down-regulated miRNA target genes were mainly involved in phenylpropanoid biosynthesis, Plant hormone signal transduction, plant-pathogen interactions and other signaling pathways. The key miRNAs were selected for NR annotation, and it was found that miR8020-x and novel-m0628-5p might be involved in the synthesis of secondary metabolites in A. sinensis vitro roots, and miR7494-y, miR477-y, miR5185-y, miR7757-y, miR408-z, and novel-m0628-5p might be responded to A. sinensis vitro roots salt stress. In this study, we used high-throughput sequencing to obtain miRNA expression profiles of A. sinensis vitro roots under salt stress, and bioinformatics analysis to obtain miRNAs that may be involved in secondary metabolites of A. sinensis and those related to the response to salt stress. The results of the present study will help to enhance the understanding of the miRNA regulatory mechanisms of A. sinensis under salt stress conditions and provide a new pathway for the synthesis of secondary metabolites of A. sinensis.