B-type natriuretic peptide (BNP) is a crucial cardiac biomarker secreted from the ventricles in response to volume and pressure overload that is used widely in the diagnosis, prognosis, and risk assessments of heart failure. Reliable and accurate measurements of BNP are essential for effective clinical decision-making. This study evaluated the analytical performance of BNP testing using an Alinity i immunoassay analyzer. The precision and linearity were assessed according to CLSI guidelines EP15-A3 and EP06-Ed2, respectively. Reference interval verification was performed in accordance with CLSI EP28-A3c using the BNP results from 20 healthy adults, and the findings were compared with the manufacturer’s reference range (<100 pg/mL). The Alinity i system showed excellent within-run and total precision, fulfilling the analytical performance specifications derived from biological variations. The linearity was maintained across the reportable range of 10~5,000 pg/mL, with a coefficient of determination (R²) greater than 0.99. For reference interval verification, 20 out of 20 samples fell within the manufacturer’s reference interval, indicating that the reference interval is suitable for use in the laboratory. These results confirm that the Alinity i BNP assay provides precise and linear results and supports its clinical applicability in heart failure evaluations. This highlights its potential for improving diagnostic accuracy and patient management through reliable BNP assessments in routine clinical settings.