Purpose The myocardial injury was induced by hypobaric hypoxia through regulating the expression of various proteins. The expression of proteins was mainly detected by western blot, and there was no systematic study on the internal control protein choice. Method The low-pressure and low-oxygen chamber was used in the study to induce myocardial injury at the altitude of 6000 meters for 24 hours and 72 hours. HE staining was used to confirm the establishment of the myocardial injury model. Western blot was used to detect the expression of different internal control proteins, including vinculin, α-tubulin, EIF5, β-actin, and GAPDH. At the same time, the total protein expression was detected by Ponceau S staining and Coomassie Blue staining. Further, the adult mouse cardiomyocytes (AMCMs) injury model was induced by hypoxia 12h and 24h. . And TUNEL staining was used to confirm the establishment of the cell injury model. Western blot was used to detect the expression of internal control proteins as same as that in vivo model. The total protein expression was detected by Ponceau S staining and Coomassie Blue staining. Result At first, the myocardial injury model was built successfully by hypobaric hypoxia 24h and 72h. The expression of internal control proteins including vinculin, EIF5, and β-actin was consistent. The expression of α-tubulin in the 24h-hypobaric hypoxia group was consistent with the control group, and the expression was significantly lower in the 72h-hypobaric hypoxia group than that in the control group. The expression of GAPDH was significantly higher in the 24h and 72h-hypobaric hypoxia groups than that in the control group. The AMCMs injury model was establised successfully by hypoxia 12h and 24h. The expression of total protein was consistent, and the expression of the internal control proteins like EIF5 and β-actin was consistent. Vinuclin, α-tubulin, and GAPDH were expressed higher in the hypoxia groups of 12 hours and 24 hours than that in the normoxic group. Conclusion: EIF5 and β-actin can be the suitable choices for internal control proteins in the study of hypobaric hypoxia-induced myocardial injury using western blot. And the total protein is also a better choice in the hypobaric hypoxia study.