Background This study aimed to assess the effect of Heart-Type Fatty Acid-Binding Protein (H-FABP), Creatinine Kinase (CK) and Homocysteine (Hcy) in determining the severity of acute poisoning. Methods 224 poisoning patients were divided into four observed groups: CO, alcohol, pesticide and drug, 50 health people as control group were included in this study. The patients were diagnosed moderate or severe acute poisoning. Blood samples were collected at the time of admission and the H-FABP, CK and Hcy of serum were tested at the time point. Results All the four observed groups of patients, the average H-FABP, CK and Hcy levels were significantly higher than those of the control group. We found H-FABP combined with CK had a statistically correlation between the moderate and severe of acute CO and pesticide poisoning, H-FABP combined with Hcy had a significantly correlation between the moderate and severe of acute alcohol poisoning, H-FABP combined with CK and Hcy had a significantly correlation between the moderate and severe of acute drug poisoning. ROC curve showed that the combination of three biomarkers could improve the diagnostic ability of the type and severity of acute poisoning. Conclusions These results indicate that changes in specific biomarkers (H-FABP combined with CK and Hcy) have potential on determining the type and the severity of acute poisoning. These findings can contribute for further research aiming a stronger model to be used in clinical practice.