Illicit administration of transgene into horses is a form of gene doping that has been a key concern in equine sports. The large number of potential performance‐enhancing transgenes has demanded a cost‐effective and reliable detection method. Multiplex qPCR is a relevant technique, but the cross‐talking between fluorophores and high background noise limits the method sensitivity and specificity. This study reports a simpler multiplexing approach by using the same fluorophore for four hydrolysis probes each targeting one of the four transgenes: human growth hormone, insulin‐like growth factor 1, equine erythropoietin and interleukin‐10. Any positive findings from this multiplex qPCR assay can then be confirmed by individual qPCR assays to identify potential transgene(s). This has effectively eliminated the cross‐talking issue and allowed an improved signal‐to‐noise than conventional multiplex qPCR assay. It has also removed the limitation imposed by the available choice of fluorophores and optical channels of qPCR instruments on the number of transgenes that can be analysed in a multiplex qPCR assay. This novel multiplex qPCR has been successfully validated. The estimated limits of detection were ~1500–2500 copies/mL of blood, thus demonstrating comparable sensitivity with the corresponding duplex qPCR assays. Concurring results were obtained by analysing hundreds of official blood samples provided by racehorses with this multiplex qPCR assay and the accredited individual duplex qPCR assays. This novel multiplex qPCR assay for detecting multiple transgenes is a cost‐effective screening method using a conventional laboratory setup and has opened up the potential to include the testing of additional transgenes in a single assay.