The growing abuse of fentanyl and its analogues (FTNs) presents a substantial public health threat, prompting the introduction of regulatory controls by government authorities. Nevertheless, existing screening strategies for FTNs are primarily based on targeted or non-targeted approaches that utilize a limited set of mass spectrometry fragmentation data, which are far from meeting the needs of class scheduling. In this study, a comprehensive non-targeted screening strategy for FTNs was developed. Firstly, existing and potential unknown structures were obtained and a database was created by combining the substitution patterns and the characteristic fragmentation routes (CFRs). Following this, a preliminary screening was executed by matching entries from the database with empirical data to identify CFRs and suggest candidate structures. The likelihood of an analyte being an FTNs increased with the identification of multiple CFRs. After that, a final judgment was made based on the similarity of the isotope distribution of the candidate structures and the spectra obtained, along with the evaluation of chromatographic peak profile. This proposed strategy could achieve an extensive coverage of 1.25 × 10