Abstract: 　Objective　The aim of this study was to evaluate the application of receiver operating characteristic (ROC) curve of biochemical indexes of blood for HbF and HbA2 on the screening and diagnosis of thalassemia in children. Methods　The traditional methods (cellulose acetate membrane electrophoresis and single one minute alkaline denaturation) as well as HPLC were used to quantitatively detecting HbF and HbA2 in 200 thalassemic cases and 62 normal controls. With DNA analysis as a golden standard for thalassemia diagnosis, ROC was applied to evaluate the indicators of HbF and HbA2 including areas under the curve (AUC), sensitivity and specificity after different cut-off values chosen. Results　In α- and β-thalassemia groups, all of AUC of HbF and HbA2 quantified by HPLC were higher than that detected by the classical method. In the β-thalassemia group, the AUC of HbA2 quantified by HPLC showed the greatest value (0.991). The coincidence rate, sensitivity and specificity of the combined screening scheme of the cut-off value HbF (≥1.05%) and HbA2 (≥3.75%) analyzed by ROC were 99.2%, 99.0%, and 100%, respectively. Conclusions　It is suggested that the combined screening scheme, the cut-off value of HbF (≥1.05) and HbA2 (≥3.75) measured by HPLC, together with the related hematological indexes, may be applied to the large-scale screening of β-thalassemia. This scheme can improve the sensitivity, coincidence rate and screening efficiency. The internal quality control for the detective process ensured the accuracy and reliability of the results, and reduced the misdiagnosis rates.