Breast cancer is a highly heterogeneous disease. Tumour expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 guides treatment decisions, but does not always predict clinical response. Tri-dimensional organoids have gained mounting interests as an advanced near-physiological model and drug screening system. We have generated organoids from 43 treatment-naïve primary breast tumours of various molecular subtypes including poor-prognostic triple-negative breast cancer. Long-term culture, expansion and cryopreservation of these organoids were achieved. We then performed a multi-omics characterisation of the established breast cancer organoids. Whole-exome sequencing analysis identified that cancer-related mutations are predominantly conserved in breast cancer organoids, although some organoid lines exhibit limited mutational similarities with the primary tumour or demonstrate evidence of a clonal drift. Both intra- and inter-molecular heterogeneities of organoid lines were identified at the single cell level. Proteomic analyses revealed that breast cancer organoids show distinct and conserved proteomic signatures between molecular subtypes. We also noticed, using immunohistochemistry, that Ki-67 staining in breast cancer organoids reflects that in primary tumours. When subjected to common chemotherapeutic drugs (doxorubicin, cyclophosphamide and paclitaxel alone or in combination), we noted a reduction in cell viability in breast cancer organoids compared to vehicle control. In conclusion, specific breast cancer organoids lines exhibit molecular features of the primary tumours. Our drug response results further highlight the potential of breast cancer organoids as a more reliable pre-clinical model in personalised medicine.