Background The bromocresol green (BCG) assay is commonly used for measuring albumin (ALB), but is affected by α₁- and α₂-globulins, which are elevated in systemic inflammation. The modified bromocresol purple (mBCP) assay is another dye-binding method developed to overcome non-specific reactions. Concentrations of α₂-macroglobulin, a major α₂-globulin component, are increased in nephrotic syndrome (NS), but not in inflammation. There is little direct evidence that α₂-macroglobulin affects BCG or mBCP assays. Methods We measured serum albumin concentrations in 33 patients with NS and 13 reference healthy controls using BCG (ALB_BCG) and mBCP (ALB_mBCP) assays, and nephelometry (nALB) as a reference method. We also determined five specific proteins belonging to the α₁- and α₂-globulins by nephelometry. After adding purified α₂-macroglobulin to albumin solutions, protein reactivity in these three assays was compared. Results Nephrotic syndrome patients were categorized to tertiles according to nALB concentration. In all tertiles, ALB_BCG was significantly higher than nALB, especially in the severe hypoalbuminemia group, in which α₂-macroglobulin was 43–49% higher. By contrast, ALB_mBCP and nALB were almost identical in all three groups. The difference between ALB_BCG and nALB was positively correlated with the α₂-macroglobulin concentration. In vitro, when α₂-macroglobulin was added to solutions containing identical albumin concentrations, α₂-macroglobulin dose-dependently increased ALB_BCG, but not ALB_mBCP. Conclusions In NS, α₂-macroglobulin is a major factor for positive bias of ALB_BCG, especially in patients with severe hypoalbuminemia. The mBCP assay is useful for measuring albumin concentrations in NS.