Cypridina luciferase, a well-known secretory enzyme involved in the bioluminescence of marine ostracod, is used to monitor gene expression in mammalian cells. Here we report the preparation of biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay (BLEIA). Recombinant Cypridina luciferase was expressed in yeast and successfully purified to near homogeneity. The luciferase was biotinylated with conventional biotinylation reagents, and the biotinylated lysine sites were determined by liquid chromatography-tandem mass spectrometry. The biotinylated luciferase was stable when stored at 4 degrees C. The stability of synthetic S-Cypridina luciferin was significantly improved by adding antioxidants to Tris-HCl buffer. The biotinylated luciferase and S-Cypridina luciferin were used in a model study of the immunoassay for interferon-alpha. The linearity of this immunoassay extended from 7.8 to 500 pg/mL interferon-alpha. Our results show that Cypridina luciferase is a very sensitive and versatile bioluminescent reporter.