The modern large-scale pyrosequencing technology is a revolution of DNA sequencing. One of the key points in this technology is to get an ATP sulfurylase immobilized on the surface of magnetic beads and with a high activity. Biotinylated ATP sulfurylase can be immobilized on magnetic beads coated with streptavidin through the specific conjunction between biotin and streptavidin, but using chemical modification method to biotinylate ATPS will affect the activity of the enzyme. ATP sulfurylase fused with the carboxyl terminal 87 residues of Escherichia coli biotin carboxyl carrier protein (BCCP87) was expressed in E. coli using fusion expression strategy. Results from Western blot analysis and SDS-PAGE analysis showed that the fusion protein could be biotinylated in vivo, and the molecular mass of the fusion protein was about 64 ku. The biotinylated ATP sulfurylase could be immobilized on the surface of magnetic beads coated with strepavidin, and the immobilized ATPS could be used for quantification of PPi and pyrosequencing. An effective enzyme for the large-scale chip-based pyrosequencing system was supplied.