MALDI-TOF mass spectrometry (MS) is powerful for the analysis and characterization of small and large molecules such as peptides, lipids and proteins. However, there is limitation in sensitivity and reproducibility specially for large molecule analysis (>10kDa), due to the lower ionization efficiency. In this study, we introduce the application of graphene layer on metal plates for MALDI-TOF MS analysis to improve ionization efficiency and reproducibility in intact protein analysis. We are developing a MALDI-TOF MS method for the detection of specific bacterial protein (i.e., KPC-2) in clinical purpose: Klebsiella pneumoniae carbapenemase (KPC-2) has a high molecular weight (~28 kDa), which is considered as antibiotics-resistant role in superbacteria. Therefore, a conventional stainless steel (SUS) plate and graphene-layer on copper plate (G/Cu) were compared for the detection and quantification of KPC-2 protein from bacteria. G/Cu plate showed lower coefficient of variation and also 6-fold higher peak intensity of KPC-2 than SUS plate. Standard curve for serial dilution series was also showed much higher linearity on G/Cu. To further confirm the performance of graphene-layer in sensitivity and reproducibility, we additionally tested several alloy plates with or without graphene-layer condition in terms of KPC-2 detection sensitivity. As expected, graphene-layer on alloy plates also showed the increase of ionization efficiency of KPC-2 protein. We concluded that graphene-layer on MALDI plates induces high intensity favorable for the intact protein detection. Finally, we suggest that graphene-layered plate is practically applicable for the identification and quantitative analysis of a target protein on MALDI-TOF MS analysis.