Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been widely applied for the analysis of large biomolecules. The emergence of inorganic material substrates and new organic matrices extends the use of MALDI MS for small molecule analyses. However, there are usually preferred matrices for different types of analytes. Here, an organic compound, 4-hydroxy-3-nitrobenzonitrile, was found to be a general purpose matrix for the analyses of small organic, peptide and protein molecules. In particular, 4-hydroxy-3-nitrobenzonitrile has a strong UV absorption property, and it provides a clean background in the low mass range. Its analytical performances as a UV-laser matrix were demonstrated for different types of analytes, including organic drugs, peptides, proteins, mouse brain tissue and bacteria. Compared with commercial matrices, this new matrix has better performances when analyzing small molecules, such as drugs, peptides and lipids, while it has similar performances when analyzing proteins.