Paraquat (PQ) poisoning remains a major public health concern in many countries. Extensive research has focused on finding early diagnostic biomarkers of acute PQ poisoning. In order to investigate the characterization of diagnostic biomarkers in PQ poisoning, we utilized proteomic analysis using serum from rats exposed to PQ, and we identified 8 differentially expressed proteins from over 500 protein spots. The expression of apolipoprotein E (ApoE), preprohaptoglobin (Pphg), a precursor of haptoglobin (Hp), and complement component 3 (C3) proteins was greatly induced by PQ exposure while the expression of fibrinogen γ-chain (FGG) and Ac-158 was dramatically reduced. To further investigate the possibility of ApoE, Pphg and FGG as useful diagnostic biomarkers of PQ poisoning, western blot and qRT-PCR analyses were conducted using cell lines as well as rat and human sera. The expression levels of ApoE, Hp and FGG were significantly altered in the presence of PQ in both rat and human serum suggesting that these proteins may be appropriate candidate molecular biomarkers for the early diagnosis of acute PQ intoxication.