In this study, we developed an integrated and automatic microfluidic immunoassay system, which comprised a multi-layer immunoassay microfluidic chip and a rotary confocal LIF scanner. The normally closed elastomeric microvalves were integrated on the multi-layer microfluidic chip to realize flexible reagents delivery for the rapid immunoassay. The various operations of immunoassay including coating, blocking and washing could be performed on a single chip with reduced reagents consumption and analysis time. The homemade rotary LIF scanner was able to detect the signals from the multichannels sequentially in a short period of time, enabling the potential capability for high-throughput immunoassay. The performance of this system was demonstrated by the sandwich immunoassay of human immunoglobulin G with a LOD < 10 ng/mL (S/N = 3). The linear range was from 500 ng/mL to 100 mu g/mL. This system has also been applied to the measurement of human immunoglobulin G from 15 real samples, demonstrating the potential of this platform for biomedical applications.