Antisense oligonucleotides (ASODNs) have been widely used as an important tool for regulating gene expression, and developed into therapeutics. Natural ODNs are susceptible to nuclease degradation, nucleic acid analogues, however, have less side effects, stronger stability and more potent activities. Large-scale de novo synthesis of a certain oligonucleotide has been very difficult and costly. In a previous preliminary study, we developed the polymerase-endonuclease amplification reaction (PEAR) for amplification and large-scale preparation of natural antisense ODNs. Here we extended the method in preparation of a widely used modified oligonucleotide with 5'-O-(1-Thiotriphosphate) modifications. Using electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection, the purity of the PEAR product was measured as high as 100.0%. Using PEAR a large amount of a specific oligonucleotide can be produced starting from a small amount of synthetic seeds. It is suggested that PEAR can be a useful tool for large-scale production of modified oligonucleotides.