Avian influenza virus H5 is a widespread virus among humans and animals which has caused fatally systemic diseases through poultry-to-person transmission in the past few years. Reverse transcription polymerase chain reaction (RT-PCR) has been proved to be an effective approach for the identification and detection of avian influenza viruses. However, conventional tube RT-PCR is slow and reagent consuming and cannot meet the need for rapid and low cost detection of pathogenic bacteria and viruses. Microfluidic PCR is a burgeoning field among the techniques based on molecular analysis. In this paper, we reported a microfluidic PCR system that integrated RT-PCR and real time fluorescence detection for rapid identification of avian influenza virus H5. This microfluidic device mainly consisted of a thermal controlling unit providing actuation for the temperature cycling needed for amplification, an optical inspection system for online recording fluorescence and a microfluidic chip fabricated using polydimethylsiloxane (PDMS). In this study, influenza virus H5 from clinical chicken throat swab specimens was rapidly detected using the RT-PCR microfluidic system, which was consistent with the results of embryonated egg culture. Compared with a large-scale device, the integrated microfluidic system presented here can perform rapid nucleic acid amplification and analysis, possibly making it a crucial platform for pathogenic bacterium and virus detection in the future.