Hemoglobin E (Hb E) is the most common hemoglobin variant in Southeast Asia. It is resulted from substitution at codon 26 of GAG to AAG. Heterozygous (Hb type: EA) and homozygous Hb E (Hb type: EE) were associated with hypochromic microcytic red blood cell and minimal anemia however compound heterozygous Hb E with β thalassemia (Hb type: EF) can cause variable severity. Hb analysis with EE or EE/EF should be confirmed in molecular analysis. Previous method, Hb E genotyping was done on AS-PCR moreover β-thalassemia large deletion with gap-PCR should be also done in another condition. Because of β-thalassemia (large deletion) with Hb E, the PCR result at codon 26 mutant will similar to homozygous Hb E. Objective of the study is to develop novel method for Hb E genotyping and β-thalassemia large deletion (3.5 kb and 45 kb) identification in single tube. We design primers related with three pair for β-globin gene codon 26, β-thalassemia 3.5 kb deletion, and β-thalassemia 45 kb deletion. The thermal cycling, melt curve and HRM analysis were performed on real-time PCR. The three specific melting curves could be difference with Tm. The amplified products were aligned according to melt curve including; Tm: 69.0 – 73.5 oC specific for β-thalassemia 45 kb deletion, Tm: 74.5 – 78.0 oC specific for β-thalassemia 3.5 kb deletion, and Tm: 78.0 – 82.0 oC for β-globin gene codon 26 fragment. Hb E genotyping was performed on codon 26 fragment using HRM analysis. The advantages of the developed method in single tube could perform around one hour, no need for a post-PCR step, avoid to ethidium bromide (mutagen). The cost effectiveness and reduced workload have more magnitude to merit the implementation in the routine setting than previous method.