Abstract: Organoid technology, a revolutionary three-dimensional stem cell culture model, can highly mimic the structure, cellular diversity, and physiological functions of human organs, offering a novel research paradigm for molecular diagnostics. Patient-derived organoids stably retain the genomic features, transcriptomic heterogeneity, and pathological phenotypes of their parental tissues during in vitro expansion and are hence recognized as “living biobanks.” This review systematically summarizes the diverse applications of organoids in molecular diagnostics. At the genomic level, they enable the identification of driver mutations and the modeling of tumor clonal evolution and drug resistance mechanisms. In clinical translation, organoid drug sensitivity testing demonstrates potential for predicting individualized treatment responses and serves as an effective platform for biomarker discovery. Furthermore, the analysis of the organoid “secretome” provides a new avenue for identifying and functionally characterizing liquid biopsy biomarkers. In the study of infectious and genetic diseases, organoid models are used to elucidate pathogen-host interaction mechanisms and to provide functional evidence for validating pathogenic mutations in rare diseases. Although challenges remain in standardization and model complexity, the integration of single-cell multi-omics, gene editing, and bioengineering technologies is expected to establish organoid technology as a core tool spanning basic research through precision clinical diagnosis and treatment, thereby laying the foundation for personalized healthcare.