Human liver organoids are expected to be a hepatocyte source for preclinical in vitro studies of drug metabolism and disposition. Although these organoids show long-term proliferation, their hepatic functions remain low. Therefore, it is necessary to enhance the hepatic functions of primary human hepatocyte (PHH)-derived organoids. Here, we propose a novel method for two dimensional (2D)-cultured hepatic differentiation from PHH-derived organoids. PHH-derived organoids were established from cryopreserved PHHs. When cultured under a 2D condition, the single cells from PHH-derived organoids were seeded on collagen type I-coated plates. Then, optimal conditions for hepatic differentiation were screened using several compounds, cytokines and growth factors. Based on the results of the screening, we determined the 2D-cultured hepatic differentiation method from PHH-derived organoids. Hepatic gene expressions in PHH-derived organoids-derived hepatocytes (Org-HEPs) were greatly increased, compared to those in PHH-derived organoids. An RNA-seq analysis showed that gene expressions related to pharmacokinetics were upregulated in Org-HEPs compared to PHH-derived organoids. The metabolic activities of CYP1A2, CYP2C8, CYP2E1 and CYP3A4 were at levels comparable to those in PHHs. We also treated Org-HEPs and PHHs with hepatotoxic drugs, such as acetaminophen, troglitazone, amiodarone and clozapine. The cell viability of Org-HEPs was almost the same as that of PHHs. These results suggested that PHH-derived organoids could be differentiated into highly functional hepatocytes in 2D culture, and Org-HEPs could be used for hepatotoxicity tests. Thus, Org-HEPs will be useful for pharmaceutical research.