To ensure fair competition and sports integrity, gene doping is prohibited in horseracing and equine sports. One gene doping method is by administering exogenous genes, called transgenes, to postnatal animals. Although several transgene detection methods have been developed for horses, many are unsuitable for multiplex detection. In this proof-of-concept study, we developed a highly sensitive and multiplex transgene detection method using multiple πCode with identification patterns printed on the surface. The following steps were employed: (1) multiplex polymerase chain reaction amplification of 12 targeted transgenes in a single tube, (2) detection using a mixture of 12 probes labeled with different πCodes, and (3) median fluorescence intensity measurement of fluorescent πCodes. Twelve transgenes cloned into plasmid vectors were targeted, and 1500 copies of each plasmid were spiked into 1.5 mL of horse plasma. Subsequently, a novel method using πCode succeeded in detecting all the transgenes using their DNA extracts. Additionally, we detected the erythropoietin (EPO) transgene in blood samples from a horse administered solely with the EPO transgene using this method. Therefore, the πCode detection method is considered suitable for multitarget gene detection in gene doping tests.