Non-target screening (NTS) based on high-resolution mass spectrometry (HRMS) is considered one of the most comprehensive approaches for the characterization of contaminants of emerging concern (CECs) in a complex sample. This study evaluated the performance of NTS in aquatic environments (including peak picking, database matching, product identification, semi-quantification, etc.) based on a self-developed data processing method using 38 glucocorticoids as testing compounds. Data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes were used for obtaining the MS2 information for in-house or online database matching. Results indicate that DDA and DIA mode have their own advantages and can complement each other. The quantification method based on LC-HRMS has shown the potential to provide a fast and acceptable result for testing compounds. Finally, a matrix spike analysis was carried out on 66 CECs across different usage categories in wastewater, surface water, and seawater matrix samples, together with a case study performed for characterizing the whole contaminants in a Pearl River sample, to better illustrate the application potential of NTS workflow and the credibility of NTS outcomes. This study provides a foundation for novel applications of HRMS data by NTS workflow to identify and quantify CECs in complex systems.