We report the development of a versatile system based on the oscillating‐flow methodology in a thermal gradient system for nucleic acid analysis. Analysis of DNA and RNA samples were performed in the device, without additional temperature control and complexity. The technique reported in this study eliminates the need for predetermined fluidic channels for thermocycles, and complexity involved with additional incubation steps required for RNA amplification. A microfluidic device was fabricated using rapid prototyping by simply sandwiching dual side adhesive Kapton tape and a polydimethylsiloxane spacer between glass microscope slides. Amplification of the 181‐bp segment of a viral phage DNA (ΦX174) and B2M gene in human RNA samples was demonstrated using the system. The developed system enables simultaneous acquisition of amplification and melt curves, eliminating the need for postprocessing. A direct comparison between the oscillating‐flow system and a commercial real‐time polymerase chain reaction (PCR) instrument showed complete agreement in PCR data and improved sample‐to‐result time by eliminating an additional 30 min melt curve step required in commercial PCR systems. An oscillating‐flow thermal gradient system for nucleic acid analysis is developed in this study. The authors reported the simultaneous acquisition of PCR data including amplification and melt information, eliminating the need for post‐processing time. A combination of a simple microfluidic channel, thermal gradient, and flow control resulted in a versatile PCR system that eliminates the need for predetermined amplification channels and additional temperature control for incubation steps. The oscillating‐flow PCR system was compared with a commercial real‐time PCR instrument.