Circulating human thyroglobulin (hTG) measurement has a pivotal role in the management of patients affected by differentiated thyroid cancer (DTC). Generally, hTG serum concentration less than 1 ng/ml is considered a marker of complete remission after total thyroid ablation. Recently, high-sensitivity immunoradiometric assays (IRMA) have been developed to detect very low hTG serum concentrations. The present study was undertaken to test a newly developed high-sensitivity hTG IRMA and to evaluate its diagnostic performance and reproducibility in the follow-up of patients affected by DTC. We retrospectively selected 156 patients without signs of recurrence and 39 patients with DTC recurrence. Serum samples were collected during L-thyroxine (T4) suppressive therapy (ONT4) and 4 weeks after T4 withdrawal (OFFT4), and hTG was measured by a specific high-sensitivity IRMA (DYNOtest Tg-plus, BRAHMS Diagnostica GmbH, Berlin, Germany). Sera showing the presence of antibodies against hTG (AbhTG) or hTG-recovery less than 80% were excluded from the study. The receiver operator characteristic (ROC) curve analysis was performed to select the best cut-off levels, and diagnostic performance of the marker was evaluated. By using ONT4 cut-off level of 0.2 ng/ml and OFFT4 cut-off level of 0.5 ng/ml we obtained a sensitivity/specificity/accuracy profile of 0.92/0.98/0.97 and 0.97/0.98/0.98, respectively. We found false-negative results in three (12%) and one (4%) out of 24 patients with cervical recurrence by using 0.2 and 0.5 ng/ml cut-off levels, respectively. However, we found false-negative results in 13 (54%) and six (25%) patients when 1.0 ng/ml cut-off level was used. Finally, DYNOtest Tg-plus showed a very satisfactory intra- and inter-assay reproducibility in the very low hTG concentration range. Based on our data, we conclude that DYNOtest Tg-plus assay is effective and accurate in evaluation of patients with DTC.