Objective CRISPR/ Cas9 technology was used to knock out glutathione S-transferase zeta 1 (GSTZ1) and fumarate acetoacetate hydrolase (FAH) in human LO2 liver cell line, to explore the effect of GSTZ1 and FAH on the growth, proliferation and migration of hepatocytes. Methods Guide RNAs ( sgRNAs) targeting GSTZ1 and FAH genes were constructed, and co-transfected with CRISPR/ Cas9 vector into LO2 cells. Three cell lines GSTZ1- / - , FAH- / - and FAH- / - / GSTZ1- / - were identified by sequencing and Western blot. The cells were used for further analysis, including the ability of hepatocyte clone formation, cell proliferation and cell migration. Results Three cell lines GSTZ1- / - , FAH- / - and FAH- / - / GSTZ1- / - were successfully established. Compared with wild-type cells, all of three edited cell lines showed higher ability of proliferation, clone formation and migration. FAH mutation has limited effect on cell phenotype, while GSTZ1 deletion greatly improved activity of the hepatocytes. The dual-gene knockout cells demonstrated moderate activity between two single mutants. Conclusions FAH/ GSTZ1 dual-mutated hepatocyte cell lines were established for the first time, which provides a new cell model for the study of tyrosine metabolism pathway and hereditary tyrosinemia type I (HT1) therapy.