BACKGROUND: Exosomes, extracellular vesicles with an average diameter of 30 ~ 150 nm, are pivotal in mediating the cellular microenvironment (CM) through their cargo-carrying capability. Despite extensive studies, the dynamic and regulatory mechanisms of exosomal cargoes, including lipids, proteins, nucleic acids, and metabolites, remain poorly understood. METHODS: In this study, we collected culture medium of ovarian cancer cells at four different time points (12, 24, 36, 48 h). Exosomes were isolated using ultracentrifugation, and miRNA sequencing was performed for exosomes from each group (T12, T24, T36, and T48). RESULTS: A total of 131 miRNAs were identified in all groups. Specifically, 41, 115, 63, and 24 miRNAs were detected in the T12, T24, T36, and T48 groups, respectively. Among these, 15 miRNAs were common to the all groups, while 3, 57, 10, and 3 miRNAs were unique to the T12, T24, T36, and T48 groups, respectively. Functional analyses of the target genes for both common and specific miRNAs indicated that numerous target genes were involved in signaling pathways and cancer-related processes. CONCLUSION: It suggested that exosomal miRNAs might be critical in intercellular communication and in dynamically remodeling the tumor microenvironment. These insights could enhance our understanding of the role of exosomal miRNAs in cancer biology and inform the development of novel therapeutic strategies.