New found in biochemical characteristics of placenta can bring new insight for further studies on the possible markers of physiological/pathological pregnancy or function of placenta. We compared the proteome of dairy cow placenta enzymatic hydrolysis by different proteases by label-free mass spectrometry approach. In total 541, 136 and 86 proteins were identified in trypsin group (TRY), pepsin group (PEP) and papain group (PAP). By comparing the proteome of PAP and TRY, 432 differentially expressed proteins (DEPs) were identified. PEP vs TRY identified 421 DEPs, while 136 DEPs were identified in PEP and PAP. The results showed the proteins identified by papain are mostly derived from extracellular matrix and collagen and enriched in relaxin signaling pathway, AGE-RAGE signaling pathway in diabetic complications; pepsin digestion can identify more muscle-related proteins, which are enriched in lysosome, platelet activation, cardiac muscle contraction, bacterial invasion of epithelial cell and small cell lung cancer; trypsin mainly enzymatically degrades extracellular matrix, blood particles, and cell surface proteins which enriched in arginine and proline metabolism, olfactory transduction proteasome, protein processing in endoplasmic reticulum, pyruvate metabolism and arrhythmogenic right ventricular cardiomyopathy (ARVC). In summary, these results provide insights into the selection of protease in dairy cow placenta proteomics.