Fluorescence-based high-throughput screening approaches facilitate the discovery of enzymes and microorganisms for polyethylene terephthalate (PET) depolymerization and recycling. However, the traditional method of activating the fluorescence signal by cleaving the ester bond on small molecule probes has limited ability in detecting enzymatic activity towards polymeric substrates. This study proposes a novel fluorescence-based screening strategy that detects the release of sulfhydryl groups during the depolymerization of pseudo-PET polythioesters by polyesterases. The strategy successfully identifies the polyester-depolymerizing activity of leaf-branch compost cutinase (LCC ^ICCG), while porcine liver esterase (PLE) only hydrolyses small molecular substrates. When combined with a droplet microfluidic system, the strategy enables high-throughput screening of LCC ^ICCG. The study also demonstrates that screening for polyester-depolymerizing bacteria can be performed via a microplate reader platform. The new screening approach offers an efficient method for identifying enzymes and microbial resources for depolymerizing polyester-like plastics.