Gene therapy, particularly lentiviral vector-based approaches, has emerged as a crucial alternative for treating β-thalassemia, one of the most prevalent monogenic disorders worldwide. This study focuses on overcoming clinical challenges associated with existing HBB lentiviral vectors, characterized by suboptimal titers and inadequate β-globin expression. We have advanced the BB305 lentiviral vector, creating a novel HBB vector, G2B, specifically engineered for β-thalassemia gene therapy. The optimization involved refining the Locus Control Region (LCR) sequence length and incorporating mutated gamma globin HGB2 promoter into the BB305 vector. Our innovative G2B vector exhibits a significant ~4-fold increase in lentiviral titer and a 50% enhancement in β-globin expression compared to the clinical BB305 vector. In the Hbbth4/Hbb+ thalassemic hematopoietic stem/progenitor cell transplantation model, the G2B vector demonstrated remarkable therapeutic efficacy without altering integration safety profiles. These findings highlight the potential of the G2B vector in treating β-thalassemia and possibly other hemoglobinopathies. Our research offers a promising pathway toward more effective and affordable gene therapy options for β-thalassemia and similar genetic disorders.