Background Fatty liver is a major metabolic disorder in dairy cows and is believed to result in major economic losses in dairy farming due to decreased health status, reproductive performance and fertility. Currently, the definitive means for diagnosing fatty liver is determining the fat content of hepatic tissue by liver biopsy, which is an invasive and costly procedure, making it poorly suited to dairy farms. Therefore, the key aim of this study was to investigate the measurement of serum paraoxonase-1 (PON1), an enzyme exclusively synthesized by the liver, as a sensitive noninvasive biomarker for diagnosis of fatty liver in dairy cows. Results A comparative cohort study using serum specimens from Holstein–Friesian dairy cows (46 healthy and 46 fatty liver cases) was conducted. Serum PON1 (paraoxonase, lactonase and arylesterase) activity and other biochemical and hematological parameters were measured. We found that serum PON1 activity was lower (P