A comparative analysis of the metabolites and metabolic pathways of fentanyl was conducted in liver microsomes and zebrafish models utilizing ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS). A total of 21 metabolites were identified across both liver microsomes and zebrafish models. These included 9 phase I metabolites, such as N-dealkylated, N-oxidated and hydroxylated products, and 12 phase II metabolites, including glucuronidated, methylated, and sulfated products, as well as a series of products derived from conjugation with glutathione. Notably, the products derived from conjugation with glutathione are reported here for the first time. This study provides a comprehensive and in-depth comparative analysis of fentanyl metabolism in liver microsomes and zebrafish, offering a foundation for analyzing and identifying biological samples in cases of fentanyl misuse and fatalities.