We have developed a novel phage display method based on catalytic activity for the in vivo selection of an enzyme. To confirm the validity of our method and to demonstrate its potential utility, we used biotin protein ligase (BPL) from Escherichia coli as a model enzyme. We were able to demonstrate the potential value of our method by selective enrichment for the birA gene, which encodes BPL, in a mixed library. The presented method for in vivo selection should allow selection of various enzymes that catalyze modification of peptides or proteins, such as protein ligase, acetylase, kinase, phosphatase, ubiquitinase, and protease (including caspase). The method should be useful in efforts to analyze mechanisms of signal transduction, to find unidentified enzymes encoded by cDNA libraries, and to exploit artificial enzymes.