Abstract Background Conventional immunoassays are labor intensive, time consuming, expensive and require large pieces of equipment for detection. In an effort to overcome these shortcomings, this study established an immunoassay method of alpha fetoprotein (AFP) in serum in combination with the microfluidic chip technology. Methods A sandwich immunoassay approach was applied to detect AFP based on microfluidic chips and the chemiluminescence as detection signal. The chip used in this method was composed of a polydimethylsiloxane (PDMS) microchannel layer over a PDMS base layer. Result AFP concentration and chemiluminescence intensity were linearly correlated over the concentration ranging from 12.5 to 200ng/ml, and a detection limit as low as 1.5ng/ml using this method. The coefficients of variation were 9.91% and 11.4% for the within- and between-run assays, respectively. More than 50 clinical samples were tested and the results obtained for this method strongly correlated with Roche's electrochemiluminescence (ECL) kit. Conclusions The proposed method offers a reliable, simple, reagent safe and inexpensive analytical platform for the determination of AFP in serum, and promotes the development of high throughput screening and point-of-care testing (POCT) diagnostics in clinical practice.