As global temperatures rise, heatstroke (HS) incidence and mortality have increased significantly, but forensic diagnostic criteria and multi-organ damage mechanisms remain unclear. In this study, a rat model of HS-induced death was established under 39 ± 0.5°C and 65% ± 5% humidity. Histopathology, ATR-FTIR spectroscopy, and machine learning identified the hypothalamus, hippocampus, liver, and spleen as the most affected organs. Transcriptomic (UID mRNA-seq) and proteomic (DIA) analyses of the hypothalamus revealed 447 differentially expressed genes and 692 differentially expressed proteins. Integrated multi-omics analysis highlighted pathways including endoplasmic reticulum protein processing, MAPK signaling, and apoptosis. Three key genes/proteins (Hspb1, Dnaja1, Jun) were validated by qRT-PCR, Western blot, and immunohistochemistry. This study provides new insights into HS diagnosis and mechanisms, offering scientific evidence for forensic and clinical applications.