Region-specific RNA modifications are crucial for advancing RNA research and therapeutics, including messenger RNA (mRNA)-based vaccines and immunotherapy. However, the predominant method, synthesizing regionally modified mRNAs with short single-stranded DNA (ssDNA) splints, encounters challenges in ligating long mRNA fragments due to the formation of RNA self-folded complex structures. To address this issue, we developed an efficient strategy using an easily obtained long double-stranded DNA (dsDNA) as a ligation splint after in situ denaturing, while parts of this dsDNA are the templates for transcribing mRNA fragments. We observed that the denatured dsDNA formed a long hybrid duplex with these mRNA fragments, overcoming their structures. Further, our novel strategy remarkably facilitated the ligation of long mRNA fragments (especially structured ones), offering ligation efficiency up to 106-fold higher than the ssDNA method. Using this one-pot strategy, we conveniently synthesized the mRNAs with N1-methylpseudouridine (m1ψ) and 5-methylcytidine (m5C) modifications in specific regions. We have found that compared with the fully modified mRNAs, the 3'UTR m1ψ modifications alone increased the translation efficiency, and the combined modifications of the m1ψ-3'UTR and m5C-5'UTR/CDS exhibited higher translation efficiency and lower immunogenicity in general. Our study presents a broadly applicable strategy for producing regionally modified mRNAs, advancing the potential of mRNA therapeutics.