Background Non-alcoholic steatoheaptitis (NASH) , the critical stage of non-alcoholic fatty liver disease (NAFLD) , is of chronic progression and can develop cirrhosis even hepatocellular carcinoma (HCC).However, non-invasive biomarkers for NASH diagnosis remain poorly applied in clinical practice.Our study aims at testing the accuracy of the combination of cytokeratin-18 M30 fragment (CK-18-M30) ,fibroblast growth factor 21 (FGF-21) ,interleukin 1 receptor antagonist (IL-1Ra) ,pigment epithelium-derived factor (PEDF) and osteoprotegerin (OPG) in diagnosing NAFLD and NASH.Methods 179 patients with biopsy-proven NAFLD were enrolled as training group,91 age-and gender-matched healthy subjects were recruited at the same time as controls.63 otherNAFLD patients were separately collected as validation group.45 alcoholic fatty liver disease (AFLD) patients,50 hepatitis B virus (HBV) patients,52 hepatitis C virus (HCV) patients were also included.Serum biomarker levels were measured by enzyme-linked immunosorbent assay.Results Serum levels of CK-18-M30,FGF-21 ,IL-1Ra and PEDF increased,while OPG decreased in a stepwise fashion in controls,non-NASH NAFLD patients and NASH patients (P < 0.01).The area under receiver-operating characteristics curve to diagnose NASH was 0.86 for CK-18-M30,0.89 for } ,0.89 for IL-1Ra,0.89 for PEDF and 0.89 for OPG.CK-18-M30 had 70% negative predictive value (NPV) and 79% positive predictive value (PPV) to diagnose NASH.A 5-step approach measuring CK-18-M30 followed by FGF21 ,IL-1Ra,PEDF and OPG gradually improved the NPV to 76% and PPV to 85% ,which reached 80% and 76% respectively in the validation cohort.Conclusion Compared to single biomarker,stepwise combination of CK-18-M30, FGF-21, IL-1 Ra, PEDF and OPG can further improve the accuracy in diagnosing NASH.