AbstractWhile fluorescence‐based high‐throughput screening (HTS) has been valuable for identifying enzymes and microorganisms for polyethylene terephthalate (PET) depolymerization, the traditional approach relies on cleaving ester bonds in small‐molecule probes, limiting its ability to directly detect activity towards polymeric PET substrates. This study proposes a novel fluorescence‐based screening strategy for detecting the release of sulfhydryl groups during the depolymerization of pseudo‐PET polythioesters by polyester hydrolases. The strategy successfully identifies the polyester‐depolymerizing activity of leaf‐branch compost cutinase (LCCICCG), whereas porcine liver esterase (PLE) only hydrolyses small molecular substrates. Furthermore, the strategy can be integrated with a droplet microfluidic system for high‐throughput screening of LCCICCG. This work further demonstrates the applicability of the microplate reader platform for screening polyester‐depolymerizing bacteria. This novel fluorescence‐based approach offers an efficient strategy to identify enzymes and microbial resources for depolymerizing polyester‐like plastics.