Abstract: Objective: To analyze the genotype of β-globin gene and try to confirm whether novel mutations existed. Methods: Blood routine analysis and hemoglobin(Hb) analysis were performed on the cases. The thalassemia genotypes were analyzed by fluorescence PCR and DNA sequencing. Results: A total of 194 cases were diagnosed with β-thalassemia, and one of them was heterozygous for a novel mutation in intron Ⅱ of the β-globin gene. The case was a two-year-old boy with mild anemia in clinical. The results of Hb analysis showed Hb A was 92.9%, with an increase in Hb A₂(4.2%) and Hb F(2.9%). DNA sequencing showed that there was a heterozygous mutation in the β-globin genes IVS-Ⅱ-561 to IVS-Ⅱ-562, involving one base deletion and 13 base insertion mutation. The genotype was identified as IVS-Ⅱ-561-562(-1 bp, +13 bp). Conclusion: The heterozygosis of the novel IVS-Ⅱ-561 to IVS-Ⅱ-562 mutation in the β-globin gene results in β-thalassemia, with mild anemia and elevated Hb A₂ levels.