Molecular cloning is one of the most important and widely used technologies in molecular biology research. Generally, the target DNA fragment and the vector are separately digested by restriction enzyme, then purified and recovered, and then ligated with DNA-ligase. For some very short gene fragments (<300 bp), the recovery efficiency of the purified fragment is very low after digestion and cleavage, leading to the difficulty in its inserting into the expression vector. To address this issue, we developed a cloning method based on restoration of antibiotic resistance in constructing recombinant plasmid, which proved highly efficient in cloning very short gene fragments.