Background Accurately evaluating liver fibrosis in patients with non-alcoholic fatty liver disease (NAFLD) is important for identifying those who may develop complications. The aims of this study were (1) to measure serum Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA + -M2BP) using the glycan sugar chain-based immunoassay and (2) to compare the results with clinical assessments of fibrosis. Methods Serum WFA + -M2BP values were retrospectively evaluated in 289 patients with NAFLD who had undergone liver biopsy. Histological findings were evaluated by three blinded, experienced liver-specific pathologists. Results For stages 0 ( n  = 35), 1 ( n  = 113), 2 ( n  = 49), 3 ( n  = 41), and 4 ( n  = 51) of liver fibrosis, the serum WFA + -M2BP cutoff indexes were 0.57, 0.70, 1.02, 1.57, and 2.96, respectively. Multivariate regression analysis showed that serum WFA + -M2BP values were associated with the stage of fibrosis (≥stage 2). The areas under the receiver operating characteristic curve (AUROC), sensitivity, and specificity of serum WFA + -M2BP were 0.876, 85.9, and 74.6 %, respectively, for severe fibrosis (≥stage 3) and were 0.879, 74.6, and 87.0 %, respectively, for cirrhosis. When compared with six non-invasive conventional markers, serum WFA + -M2BP had the greatest AUROC for diagnosing severe fibrosis and cirrhosis. Conclusions Serum WFA + -M2BP values are useful for assessing the stage of liver fibrosis in patients with NAFLD.