Dengue virus (DENV) is the causative agent of one of the most important febrile illnesses worldwide. Four closely related serotypes of DENV have been described as responsible for a broad clinical spectrum of the disease. For more than a decade, molecular methods such as RT-PCR and more recently real-time RT-PCR have become important in serotyping because of their high sensitivity and specificity. For the validation of molecular test results, positive controls are required, consisting of equimolecular mixtures of viral RNA from each of the four serotypes commonly obtained from in vitro cell cultures. In this study, the in silico design of a plasmid with the target sequences to the oligonucleotides and probes for DENV serotyping from Johnson et al (J Clin Microbiol. 2005; 43:4977-83) was performed, to generate a multi-target control RNA. The plasmid pBluescript II KS (+) with the cloned DENV target sequences, was obtained by commercial gene synthesis service, propagated by transformation of E. coli DH5α and verified by restriction analysis. The plasmid was linearized by enzyme digestion and used to obtain large amounts of RNA by run-off in vitro transcription. The transcripts were treated with DNase I, purified and used as a positive control in the serotyping of DENV by qRT-PCR. The control RNA was used successfully as a positive control in the serotyping and quantification of DENV from clinical isolates, with a high control RNA yield from a single in vitro transcription reaction.