Abstract Objective The occurrence of non-alcoholic fatty liver disease (NAFLD) is globally increasing. To challenge the current incidence of NAFLD, non-invasive markers that could identify patients at risk or monitor disease progression are an important need. Copper intake and organ copper concentrations have earlier been linked to NAFLD progression, but serum copper does not adequately represent the disease state. Cu atoms occur under the form of two stable isotopes, ⁶³Cu and ⁶⁵Cu, and the ratio of both (expressed as δ⁶⁵Cu, in ‰) in blood serum has been shown to be altered in chronic liver disease. To assess whether the Cu isotope ratio might predict disease occurrence and progression of NAFLD, the serum Cu isotopic composition of patients with different stages of NAFLD was determined. Results Our results showed that serum δ⁶⁵Cu values were lower in NAFLD patients, already at the level of simple steatosis, and remained stable during further disease progression. ROC analysis shows an almost perfect diagnostic ability of serum δ⁶⁵Cu values for NAFLD, but no discrimination between different severity degrees could be made. Therefore, the serum Cu isotopic composition might show potential for early diagnosis of NAFLD patients.