Phospholipids are essential for various cellular processes. Their primary role is to form a barrier around cells and organelles through a phospholipid bilayer.1 Phosphatidic acid (PA) is the simplest naturally occurring glycerophospholipid in living organisms.2 Although it represents only ~1% of total membrane phospholipids, PA plays crucial roles in numerous cellular processes, including vesicular trafficking, signal transduction, and cell proliferation.3 Given its functional significance, understanding how PA distribution and localization change over time within living cells is essential. This requires the development of fluorescent sensors capable of selectively recognizing PA over other membrane phospholipids. However, achieving such selectivity remains challenging due to the structural similarities among phospholipids. In this study, we report the design of a peptide-based fluorescent probe incorporating a zinc (Zn(II))–coordinated 8-hydroxyquinoline moiety, which exhibits selective binding to PA-containing phospholipids. The probe displays a strong fluorescence turn-on response toward PA-containing liposomes, with ~8-fold selective turn-on response over membranes containing other anionic phospholipids. We also found that membrane properties strongly affect probe binding. Changes in acyl chain saturation, phosphatidylcholine composition, membrane curvature, and pH altered the fluorescence response, consistent with differences in PA accessibility and charge state.