Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease-mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp-25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into Escherichia coli to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments.