Gene synthesis is gaining popularity as a versatile means of cloning genes. The prevailing synthesis methods are PCR-based and ligase-based gene synthesis. Both of them are not independent of the sequence content of the target gene. In the present study, a new gene synthesis method—isothermal unidirectional elongation method (IUEM) is proposed, which involves the cooperation of three enzymes, and is independent or partially independent of the target gene sequence by the specially designed oligonucleotides that form hairpins. The synthesis process is isothermal and serial in IUEM. Several factors that affect the elongation were studied, and two DNA segments of length 254 bp and 300 bp were synthesized with IUEM. The synthesized segments could work as building blocks for the synthesis of longer DNA sequences.