Due to its super sensitivity and incomparable high throughput, fluorescence-activated cell sorting (FACS) based screening method is emerging as an important ultrahigh-throughput enzymatic screening technology recently. It could detect multiple fluorescent parameters simultaneously and screen enzyme libraries at an extremely high speed (>10⁸/d), thus possesses many advantages over the conventional technologies. FACS based screening method greatly improves our ability in handling large protein libraries and has tremendous potential in various fields such as metagenomics and directed evolution. Herein, recent progress of FACS based enzymatic screening method has been reviewed, with an emphasis on its applications in enzyme directed evolution.