The potential environmental risks of glucocorticoids (GCs) have attracted attention in the past few decades. In this study, a novel diffusive gradients in thin-films (DGT) device and analytical technique based on the second generation of polar enhanced phase (PEP-2), PEP-2-DGT, were developed for sampling and quantifying natural and synthetic GCs in aquatic systems. The capacity of PEP-2 gels for accumulating all target compounds was >600 μg per disc, sufficient for long-term passive sampling of selected GCs, even in wastewaters. Systematic tests were carried out to verify the application of DGT in natural waters and wastewaters. The performance of PEP-2-DGT devices was independent (CDGT/Csoln was in the acceptable range of 0.9–1.1) of a wide range of environmental conditions: ionic strength (0.001–0.5 mol L–1), dissolved organic matter (0–20 mg L–1), and pH (3.06–9.02). It was tested for various diffusive layer thicknesses (0.565–2.065 mm) and different deployment times (10–168 h). Diffusion coefficients (D) of selected GCs through an agarose-based diffusive gel were determined for the first time (3.80–4.85 × 10–6 cm–2 s –1 at 25 °C). Linear correlations between D and log Kow were established for three groups of target GCs (R2 = 0.96–0.99). This could enable prediction of D values for other GCs with similar structures in the future, which will help for rapid screening and emergency monitoring. Concentrations and distribution patterns of analytes obtained by PEP-2-DGT devices in five rivers after 7- and 14-day deployments were in accordance with those measured from grab samples, with total GC concentrations ranging from 7 to 27 ng L–1 at all sampling sites, confirming the reliability and robustness of the DGT devices for monitoring GCs in natural waters. The development of the new DGT technique will help improve understanding of the behavior and fate of these compounds in the aquatic environments.