This letter reports a chemiluminescene immunoassay method combined with immunomagnetic separation to rapidly detect Cyfra 21-1, in which bio-functionlized magnetic nanocomposites were used as mobile substrate for capturing and isolating the cyfra 21-1 proteins. After the captured Cyfra 21-1 further reacted with horseradish peroxidase-conjugated anti-Cyfra 21-1 antibody to form a sandwich immunocomplex, the chemiluminescence would be produced as a result of addition of the chemiluminescent substrate. A home-made optical biosensor was designed to detect the chemiluminescence instead of other large instruments. There is a good linear response between the chemiluminescence intensity and the concentration of Cyfra 21-1 in the range from 0.2 to 50 ng/mL. The whole detection process including incubation, washing and detection could be performed within 45 min. The proposed method offers a simple, noninvasive and reliable tool for detecting non-small cell lung cancer and has potential application for clinical testing.