Phospholipase A (PLA) is a membrane lytic enzyme that is present in many organisms. Human PLA has emerged as a potential biomarker as well as a therapeutic target for several diseases including cancer, cardiovascular diseases, and some inflammatory diseases. The current study focuses on the development of lipo-beads that are very reactive and highly sensitive to PLA. To develop the best supported lipid bilayer formulation, several lipid combinations were investigated using 10 μm porous silica beads. The reactivity of PLA was monitored via the decrease in particle fluorescence because of the release of entrapped fluorescent dye from the particle pores or the disintegration of a fluorescent lipid constituted on the bilayer upon lipid hydrolysis using flow cytometry. The enzyme binding studies indicate that lipo-beads with bulky fluorescent tags in the lipid head group and anionic lipids produce a more pronounced response. The kinetic studies suggest that these lipo-beads are very reactive with PLA and can generate a detectable signal in less than 5 min. The enzyme inhibition studies were also conducted with two known PLA inhibitors, varespladib and quercetin. We find that quercetin can hydrolyze the supported membrane, and thus inhibition of PLA is not observed; however, varespladib has shown significant PLA inhibition on lipo-beads. We have demonstrated that our lipo-bead-based approach can detect annexin-3, a known disease biomarker, as low as 10 nM within 5 min after incubation.