<p>Diagnosis of death due to electrocution is difficult when typical electric marks are absent. A valid diagnostic method based on useful biomarkers are needed for these atypical electrocution cases. Herein we utilized formalin-fixed paraffin-embedded samples that are archived in our forensic center to conduct label-free proteome analysis in the aim of screening differentially expressed proteins (DEPs) for diagnosis of atypical electrocution. By using cases dying from mechanical injury and drug intoxication as negative controls, a total of 1591 proteins were identified, among which 128 proteins (8.0%) were DEPs to the atypical electrocution cases. These DEPs were mainly enriched in processes involved in cellular metabolism, DNA replication and gene transcription. The vast majority of the DEPs (121/128, 94.5%) were downregulated, while the remaining DEPs were upregulated in the hearts from atypical electrocution. Mitofusin-2 (MFN2) and Nucleolin (NCL) were the top upregulated and downregulated DEPs, respectively, that showed the greatest fold-changes. Immunohistochemical staining in independent case series verified that both MFN2 and NCL altered significantly in heart specimens from atypical electrocution death. Cardiac expression of MFN2 and NCL were not affected by decedents’ age, sex, or postmortem interval. Receiver operating characteristic (ROC) curve analysis revealed that the areas under the curve (AUC) of the two proteins were 0.8925 and 0.9008, with diagnostic sensitivity being 87.54% and 88.89%, and specificity being 74.19% and 84.12%, respectively. Our results suggest that cardiac MFN2 and NCL are potential useful biomarkers for forensic diagnosis of atypical electrocution death.</p>