Recently, combination method of the Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR), called one-step RT-qPCR has emerged as a powerful method for analysis of RNA targets. Especially, since the one-step RT-qPCR requires minimal sample handling and only needs brief workflow, it has significant merit for point-of-care analysis. However, this method has critical limitation, which is ‘severe side products’. Presence of PCR primers at the RT process induces major portion of side products. To solve this problem, we utilized thermo-responsive polymer as a capsule containing PCR primer. The thermo-responsive capsule was maded from commercially available Upper Critical Solution Temperature (UCST) materials, which has critical temperature above the RT thermal condition. The PCR primers inside the capsule could be isolated during RT process, so the side product generation was completely prevented. For the subsequent PCR process, the capsule was melted and the contained PCR primers could be released out and participate into PCR effectively. Using this thermo-responsive material based delivery system, we enhanced sensitivity 10 times more than conventional methods with quantitative stability and achieved reaction efficiency over 90%. Moreover, we presented mutliplex one-step RT-qPCR, which has been avoided previously due to its severe side product problem. Influenza viruses were individually identified within 30 minutes. This novel and powerful method will be utilized for many other nucleic acid targets, because it is not limited to specific target, but has general versatility for general RNA and DNA.