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The 5-year survival rate of patients with locally advanced and metastatic pancreatic cancer is less than 3.0%. Characterized by insidious early onset and high malignancy, most patients miss the optimal treatment window upon diagnosis, leading to persistently high overall mortality. Accordingly, there is an urgent clinical demand to establish efficient early diagnostic systems and develop novel targeted therapeutic strategies. More than 95% of pancreatic malignancies are of exocrine origin, mainly derived from pancreatic acinar cells and ductal epithelial cells. To date, the cellular origin of pancreatic ductal adenocarcinoma (PDAC) has not been fully elucidated. Abnormal expression and dysfunction of key genes including KRAS, p16INK4A/CDKN2A, TP53 and SMAD4/DPC4 represent core molecular events driving the malignant progression of PDAC. As an innovative in vitro research platform, tumor organoids provide powerful experimental support for in-depth dissection of pathogenic mechanisms of pancreatic cancer and the development of innovative therapeutic regimens. Human pancreatic cancer organoids are established via three-dimensional in vitro culture using clinical tumor tissues or malignant effusion specimens obtained from patients. This model can faithfully recapitulate the cytological features, histopathological morphology and molecular profiles of primary tumors, maximally preserving the authentic biological traits of tumors and effectively overcoming the limitations of conventional research models. Pancreatic cancer organoids possess diverse application values in basic tumor research and clinical translation, as summarized below: Precisely simulating tumor heterogeneity and the complex microenvironment of pancreatic cancer, providing a stable and reliable in vitro model for systematically investigating the molecular mechanisms underlying tumor initiation, malignant progression and distant metastasis. Patient-derived organoids can steadily maintain the inherent biological phenotypes and genetic characteristics of primary tumors during long-term in vitro culture. High-throughput drug screening and individualized therapeutic evaluation: Drug sensitivity and cytotoxicity assays based on organoid models yield results highly consistent with actual clinical treatment responses, enabling accurate prediction of patient prognosis and offering experimental evidence for formulating individualized clinical regimens. Combined with CRISPR/Cas9 gene-editing technology, specific gene knock-out and knock-in modifications can be performed on pancreatic organoids, providing technical support for exploring the biological functions of key genes and screening novel anti-tumor therapeutic targets. Establishing co-culture systems of tumor organoids with immune cells, fibroblasts and other stromal cells enables faithful reconstruction of the complex in vivo tumor microenvironment. Such systems help clarify the interaction mechanisms between tumor cells and stromal cells, uncover core mechanisms of tumor immune evasion, and facilitate the research, development and clinical translation of innovative tumor immunotherapeutic strategies.