Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a simple and fast technique for the analysis of large biomolecules but is not suitable for the detection of low molecular weight molecules and compounds, such as flavonoids and phenylpropanoids, mainly due to the lack of an appropriate matrix. Flavonoids and phenylpropanoids, such as coumarin and its derivatives, have attracted much attention recently because of their pharmacological activities and putative therapeutic benefits. In this study, we developed a quick and simple LDI-TOF MS method for the detection of flavonoids and the derivatives of coumarin. Analytes were spotted onto a matrix of graphene-based nanoparticles and then analyzed by LDI-TOF MS in the negative ion mode. Analysis of the sensitivity and effect of different graphene-based nanoparticles including graphene, graphene oxide, and reduced graphene oxide on desorption/ionization of analytes showed that graphene oxide was the most suitable matrix. Moreover, we found that graphene oxide sheets of larger lateral size resulted in better desorption/ionization efficiency. Overall, we show that graphene oxide is a useful matrix for the analysis of flavonoids and the derivatives of coumarin by LDI-TOF MS in the negative ion mode.