Telomere-specific quantitative fluorescent in situ hybridization (Q-FISH) accurately evaluates hepatocellular aging on histological sections, but it requires appropriate tissue processing. To establish a more simple method for the assessment of hepatocellular aging, the usefulness of nuclear size measurement was clarified using biopsy liver samples from 64 patients with non-alcoholic fatty liver disease (NAFLD), a model for oxidative stress-associated hepatocellular aging, and 11 control individuals. Relative telomere intensity (RTI) was measured on Q-FISH, and the relative nuclear size (RNS) was calculated as the average nuclear size of the hepatocytes divided by that of lymphocytes. In normal individuals and NAFLD patients, the RTI and RNS were negatively correlated. The degree of nuclear enlargement in NAFLD patients was larger than that in normal individuals with the same telomere length, possibly reflecting telomere-independent senescence. In NAFLD patients with RNS >2.0, the regenerative responses, indicated by the ratio of Ki-67-positive index to serum alanine aminotransferase level, were significantly reduced. The RNS positively correlated with the p21 expression, another marker of senescence. This all indicates that nuclear enlargement progresses in parallel with reduced regenerative responses, telomere shortening, and p21 upregulation. Nuclear size measurement is an effective method for estimation of hepatocellular aging.