Objective To explore the culture of a hepatocellular carcinoma organoid for drug screening, providing clinical reference for individualized treatment. Methods The tumor tissue from a hepatocellular carcinoma patient was digested into single cells and inoculated into Matrigel for culture. The cell morphology of the organoid and primary tumor tissue was observed by hematoxylin-eosin (H-E) staining. Immunohistochemistry staining (IHC) was used to verify the expression of cytokeratin 7 (CK7), cytokeratin 8 (CK8), p53, and glypican 3 (GPC-3) in the organoid and primary tumor tissue; the mutations of tumor-related genes in the organoid were detected by targeted sequencing. Drug screening was carried out according to the targeted sequencing results. The effects of varlitinib (a specific inhibitor of epidermal growth factor receptor/erb-b2 receptor tyrosine kinase 2[ERBB2]), sorafenib (a multi-targeted inhibitor of kinase), cisplatin and nutlin3 (a p53 activator) on the organoid viability were detected by CellTiter-Glo luminescent cell viability assay. Results The hepatocellular carcinoma organoid with tumor protein p53 (TP53) and ERBB2 gene mutations was successfully established.H-E staining showed that the structural and cytological characteristics were very similar between the organoid and primary tumor tissue. IHC staining showed that CK7 was negative in the organoid and primary tumor tissue, while CK8, p53, and GPC-3 were positive. Targeted sequencing results showed that the mutation genes in the organoid were TP53 (E271K, 100%), ERBB2 (A1232V, 62.29%), RUNX family transcription factor 1 (RUNX1) (E395A, 20.21%), and androgen receptor (AR) (Q91dup, 70%). The results of the cell viability assay showed that the half inhibitory concentration (IC₅₀) values of varlitinib and sorafenib on the organoid were 2.81 μmol/L and 1.327 μmol/L, respectively; the IC₅₀ of cisplatin was 40.98 μmol/L; while nutlin3 had no significant effect on the organoid. Conclusion We have successfully established a hepatocellular carcinoma organoid with TP53 and ERBB2 mutations. This organoid retains the structural and molecular characteristics of the primary tumor tissue. The results of drug screening may provide a reference for the individualized treatment.