The newly developed Axis-Shield clinical chemistry heparin-binding protein (HBP) assay (Axis-Shield Diagnostics Ltd., Dundee, Scotland) can be applied to fully automated platforms. We aimed to establish a reference interval (RI) of HBP using the Axis-Shield HBP assay, and to evaluate the analytical performance of this assay. An RI was established in 212 sodium citrated plasma samples using the non-parametric method (2.5th and 97.5th percentiles). Precision, linearity, and carry-over were evaluated according to the Clinical and Laboratory Standards Institute guidelines. The RI of HBP was between 5.3 ng/mL and 171.0 ng/mL, which could be applied regardless of gender and age. Percentage coefficients of variations (%CVs) of repeatability and within-laboratory precision were 4.9% and 6.3%, respectively, for low-concentration control and 1.6% and 3.0%, respectively, for high-concentration control. The linearity was excellent (coefficient of determination (R2) = 0.99), and the carry-over rate was negligible (0.05%). This is the first study to establish an RI of HBP using the newly developed and fully automated Axis-Shield HBP assay. The Axis-Shield HBP assay showed an acceptable level of analytical performance and could be used to measure HBP concentrations effectively in routine clinical practice. Further studies are awaited to evaluate the clinical utility of HBP using this automated assay.