Despite various roles of phosphatidic acid (PA) in cellular functions such as lipid homeostasis and vesicular trafficking, there is a lack of high-affinity tools to study PA in live cells. After analysis of the predicted structure of the LNS2 domain in the lipid transfer protein Nir1, we suspected that this domain could serve as a novel PA biosensor. We created a fluorescently tagged Nir1-LNS2 construct and then performed liposome binding assays as well as pharmacological and genetic manipulations of HEK293A cells to determine how specific lipids affect the interaction of Nir1-LNS2 with membranes. We found that Nir1-LNS2 bound to both PA and PIP2 in vitro. Interestingly, only PA was necessary and sufficient to localize Nir1-LNS2 to membranes in cells. Nir1-LNS2 also showed a heightened responsiveness to PA when compared to biosensors using the Spo20 PA binding domain (PABD). Nir1-LNS2's high sensitivity revealed a modest but discernible contribution of PLD to PA production downstream of muscarinic receptors, which has not been visualized with previous Spo20-based probes. In summary, Nir1-LNS2 emerges as a versatile and sensitive biosensor, offering researchers a new powerful tool for real-time investigation of PA dynamics in live cells.