Post-mortem diagnosis of fatal hypothermia (FHT) is challenging in forensic practice because traditional morphological and biochemical methods lack specificity. Recent studies have reported that brown adipose tissue (BAT) is activated during cold-induced non-shivering thermogenesis in mammals, but BAT has not been used to diagnose FHT. The aim of this study was to identify novel biomarkers in BAT for FHT based on morphological changes and differential protein expression. Two FHT animal models were created by exposing mice to 4 or -20 °C at 50% humidity. Morphologically, the unilocular lipid droplet content was significantly increased in BAT of FHT model mice compared with that of control mice. Proteomics analysis revealed a total of 283 and 266 differentially expressed proteins (DEPs) between the 4 or -20 °C FHT subgroups and control group, respectively. In addition, 140 proteins were shared between the FHT subgroups. GO and KEGG analyses revealed that the shared DEPs were mainly enriched in pathways associated with metabolism, oxidative phosphorylation, and thermogenesis. Further screening (|log2FC| > 1.6, q-value (FDR) < 0.05) identified GMFB, KDM1A, DDX6, RAB1B, SHMT-1, CLPTM1, and LMF1 as candidate biomarkers of FHT. Subsequent validation experiments were performed in FHT model mice using classic immunohistochemistry and western blotting. RAB1B and GMFB expression was further verified in BAT specimens from human cases of FHT. The results demonstrate that BAT can be used as a target organ for FHT diagnosis employing RAB1B and GMFB as biological markers, thus providing a new strategy for the post-mortem diagnosis of FHT in forensic practice.