The design, preparation and performance for novel UV-light absorbing (room-temperature) ionic liquid matrices (UV-RTILMs) for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) were reported. A series of UV-RTILMs was prepared by ultrasonication of equimolar of acid (mefenamic acid) and bases (aniline (ANI), pyridine (Pyr), dimethyl aniline (DMANI) and 2-methyl picoline (2-P)). The UV-RTILMs have not only significant absorbance at the desired wavelength (337 nm of the N2 Laser), but also have available protons that can easily undergo proton transfer reactions to ionize the target molecules. The novel UV-RTILMs have the ability to ionize different and wide classes of compounds such as drugs, carbohydrate, and amino acids. The new UV-RTILMs series have been successfully and selectively applied for biosensing the lysates of pathogenic bacteria in the presence of the cell macromolecules. A new strategy for biosensing pathogens was presented via sensing the pathogens lysate in the cell suspension. The new materials can effectively detect the bacterial toxins without separation or any pretreatment. They offered excellent ionization of labile oligosaccharides with protonated peaks. They could significantly enhance the analyte signals, produce homogeneous spotting, reducing spot-to-spot variation, excellent vacuum stability, higher ion peak intensity, and wide application possibility. The physical parameters such as molar refractivity, molar volume, parachor, surface tension, density and polarizability were calculated and tabulated. The new UV-RTILMs could offer excellent reproducibility and great repeatability and they are promising matrices for wide applications on MALDI-MS.