With purpose of performing biotinylation of firefly luciferase in vivo, we fused a fragment of gene encoding C-terminal 87 amino acids of biotin carboxyl carrier protein (BCCP) of E.coli with luciferase cDNA from firefly Pyrocoelia pectoralis. Biotin was covalently linked to a specific lysine residue of BCCP via catalysis of biotin holoenzyme sythetase in E.coli, and firefly luciferase was biotinylated indirectly as a result of fusion with BCCP. Advantages of specific coupling between biotin and avidin or streptavidin allow firefly luciferase to be immobilized on solid support coated with avidin or streptavidin, so that more flexible and convenient methods of luminescence assay will be available in application. The details of cloning, expression and function of biotinylated luciferase will be discussed in this paper.