In animals, beta-carotene 15,15'-monooxygenase (BCMO) is the key enzyme involved in the metabolism of plant beta-carotene to retinal. In the present study, we utilized beta-carotene-producing Escherichia coli to screen for mutants with higher BCMO activity which was monitored by color changes derived from beta-carotene cleavage. Recombinant wild-type and T381L mutant BCMO proteins were purified to near homogeneity in E. coli, and their enzymatic activities were determined by HPLC analysis. The catalytic efficiency for beta-carotene and retinal production of the mutant were 1.5-fold and 1.7-fold higher than those of wild-type, respectively. Further BCMO function in mammalian cells was analyzed by a retinoic acid receptor reporter assay, which responds to the metabolic conversion of beta-carotene to retinoic acid in vivo. Overall, these tools can be used to screen more active BCMO for the industrial and pharmacological purpose of retinal production from beta-carotene.