omega-Transaminase (omega-TA) is an attractive alternative to metal catalysts for the stereoselective amination of prochiral ketones. The narrow substrate scope of an R-omega-transaminase from Mycobacterium vanbaalenii (MvTA) limits its application in R-amine synthesis. A fluorescence-based TA activity screening system was developed to extend its substrate scope. The reactions were conducted in microtiter plates (MTPs) and displayed low background interference, high sensitivity (mu M magnitude), and a wide dynamic range (-factor > 0.9). A KnowVolution campaign was performed on this enzyme, and screening similar to 8000 clones with this fluorescence-based screening system resulted in two beneficial substitutions (G68Y and F129A) and three improved variants (M3, M4, and M5). The best variant, MvTA M5 (WT+G68Y+F129A), achieved the highest catalytic efficiency (toward fluorogenic substrate NMA) which was 3.2-fold higher than that of the WT enzyme. MvTA M5 exhibited significantly enhanced activity toward six different prochiral ketones with e.e. > 99% (R). The specific activity of MvTA M5 was more than 100 times higher than that of the WT enzyme toward acetonaphthone (M5: 8.1 U/mg, WT: similar to 0.07 U/mg), and it showed the highest activity on acetonaphthone, p-ethylacetophenone, and phenylacetone.