β-thalassemia is an autosomal recessive monogenic disease that is caused by defects in the production of β-like globin chains. Activation of γ-globin gene and the increase in fetal hemoglobin expression have been demonstrated as one of the most important factors to ameliorate the clinical outcome of β-thalassemia patients. In this study, 202 genes or miRNAs associated with human hemoglobin gene expression from 1802 β-thalassemia patients were analyzed with target capture and next generation sequencing strategies in terms of functional variants that might affect hemoglobin gene expression. The subsequent bioinformatics analysis included assessments of sequence quality, the variants within the target regions and the 5′UTR with potential effects on upstream open reading frames (uORFs). Among the 41 variants in 5′UTR potentially affecting the uORFs identified in the study, two variants (chr19: 41859418 G>A and chr1:153606541 C>T) were experimentally validated with dual-luciferase assays to be capable of significantly down-regulating the expression of TGFB1 and CHTOP gene, respectively. The present study demonstrated a system suitable for evaluating the importance of variants in 5′UTRs affecting uORFs in 202 human genes associated with hemoglobin expression. Research with this approach could provide potential targets that may contribute to the clinical phenotypes and provide biomarkers for precise diagnosis of β-thalassemia.