Measurement of free plasma metanephrines (metanephrine and normetanephrine), usually performed by high-performance liquid chromatography with electrochemical detection (HPLC-ECD), has been recommended as the single biochemical test of choice for the diagnosis of pheochromocytoma. Alternatively, a widely available, simple means to measure these biomarkers with enzyme immunoassay (EIA) needs to be studied. The aim of this study was to investigate the diagnostic efficacy of such a method in comparison with 131I-metaiodobenzylguanidine (MIBG) whole body scan (WBS) in patients with pheochromocytoma. We enrolled patients undergoing 131I-MIBG WBS due to clinical findings suggestive of pheochromocytoma (n = 45), and patients with primary hypertension (n = 36). All subjects had blood tests for free plasma metanephrine (MN) and normetanephrine (NM) with a commercially available EIA kit. WBS was positive in 30 pheochromocytoma patients and negative in 15 refuted ones, with 100% accuracy. The sensitivity, specificity and accuracy of MN and NM in combination (either or both positive) were 96.7%, 86.3% and 90.1%, showing comparable diagnostic performance both to 131I-MIBG WBS (all p > 0.1), and also to the same markers measured with HPLC-ECD reported in the literature. These results showed that the EIA method may be eligible as an alternative to HPLC-ECD for plasma metanephrine determination in the identification of pheochromocytoma.