Pools of short synthetic oligonucleotides (oligos) are required in the multiplex and parallel DNA construction. Microarray technology provides a fast and economical mean for massive parallel synthesis of oligos. The method of oligo synthesis with the programmable microfluidic PicoArray could simultaneously synthesize the designed oligos for multiple riboswitch genes. The synthetic oligos were recovered and purified as a pool of oligo mixture (OligoMix). Three temperature steps were employed to denature, anneal and extend the designed OligoMix until, after multiple rounds of thermocycling, the riboswitches with the desired length are obtained. The OligoMix was amplified using this PCR-based technique and the flanking adapter segments were cleaved for following assembly. Based on these oligos derived from 197 riboswitch sequences, the method of simultaneous assembling multiplex riboswitches (SAMRs) showed high fidelity by sequence identification. The resultant error rate was determined to be 2.78 parts per thousand. With the templates from SAMRs, in vitro transcription was applied to produce milligram amounts of biologically active riboswitches. With the verification of biological activity based on the high specificity of recognizing small-molecule metabolites as well as the DNA sequence redivivus by RT-PCR, the assembled riboswitches can be used for further gene operation and biological application. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1228-1235, 2009