Detection of tumor marker is an effective and low cost means in the early diagnosis of lung cancer, especially multiple tumor markers combined can improve the diagnostic value. In this paper, a chemiluminescence immunoassay using bio-functionlized magnetic nanocomposites (BFMNs) was proposed for simultaneous detection of multiple tumor markers. Carcinoembryonic antigen (CEA), neuron specific enolase (NSE) and cytokeratin 19 fragment (CYFRA21-1) were chose as the detection models. BFMNs were prepared in advance by loading different antibodies to magnetic beads containing Fe2O3 nanoparticles, and characterized using a scanning electron microscope. The BFMNs could specifically capture the corresponding antigen and its HRP labeled antibodies based on a sandwich-type immunoreaction. The immunocomplex further reacts with chemiluminescent substrate and the produced chemiluminescences were detected by a homemade three-channel luminometer. Preliminary results show that there is a good linear relationship between the chemiluminescence intensity and the CEA concentration in the range from 5 to 200 ng/mL, as well as with similar results for the detection of NSE and CYFRA21-1. The whole detection process, including the immune response, the washing and the optical signal detection steps, could be performed to carry out simultaneous detection of three tumor markers within 35 min. The proposed method offers a simple, noninvasive and rapid tool for detection of multiple tumor markers and has potential application in clinical testing.