Chromatin accessibility is an important feature of cis-regulatory elements that shapes gene regulation in plants; however, it has been studied less frequently in traditional medicinal plants such as Gardenia jasminoides J.Ellis (G. jasminoides). This study applied Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) technology to characterize the genome-wide distribution and functional features of accessible chromatin regions (ACRs) in G. jasminoides leaves. A total of 26,461 ACRs and 13,625 associated genes were identified in this study, and our results revealed a positive correlation between the open chromatin state of ACRs and the expression levels of their associated genes. These ACRs were also found to be enriched with numerous conserved transcription factor binding motifs (TF motifs). Integration of two histone modification datasets further demonstrated that ACRs are closely associated with activating histone modifications, including H3K4me3 and H3K27ac, which work together to regulate the transcription of target genes. Additionally, luciferase (LUC) reporter assays validated the transcriptional activation activity of two candidate ACRs. Taken together, these findings elucidate the genomic features of ACRs in G. jasminoides and provide critical genomic resources for subsequent gene regulation analyses for this species.