Application of two new series of ionic liquid matrices (ILMs) based on the two most predominantly used conventional organic matrices (Sinapinic acid and 2,5-DHB) in conjugation with various bases (aniline (ANI), dimethyl aniline (DMANI), diethylamine (DEA), dicyclohexylamine (DCHA), pyridine (Pyr), 2-picoline(2-P), 3-picoline(3-P)) for bacterial analysis in matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS) are reported. The results reveal that ionic liquid matrices could significantly enhance the protein signals, reduce spot-to-spot variation and increase spot homogeneity. More importantly, these novel matrices would not produce any interference during MALDI-MS analysis. Among these ILMs, 2,5-DHB/ANI, 2,5-DHB/DMANI and 2,5-DHB/Pyr can be successfully applied to intact bacterial studies compared with other ILMs. Base molecules when added to conventional matrix can promote proton transfer between the bacterial lysate and the matrices. Due to the enhanced proton transfer efficiency by the ionic liquid matrices, almost all the biomolecules of the intact bacterial cells can be ionized and detected in the MALDI-MS. All synthesized ILMs were characterized using ESI (+)/MS and UV-spectroscopy.