Abstract: Membrane microdomains, which are highly dynamic structures rich in sterols and sphingolipids on the plasma membrane, play a crucial role in various biological processes such as signal transduction, vesicle transport, endocytosis, and exocytosis. Consequently, the investigation of membrane microdomain dynamics stands as one of the important areas of research in plant cell biology. Fluorescence probes combined with fluorescence microscopy are widely used to monitor the status of living plant cells. PA probe (push-pull pyrene) is a novel, highly efficient and stable fluorescence probe based on pyrene, however, its application in imaging studies of living plant cells is limited. In this study, we used PA probes and Laser scanning confocal microscopy, combined with image processing and polar normalized value mapping method, to quantitatively analyze the order of the plasma membrane in Arabidopsis root cells. The results showed that the emission spectrum of the liquid-ordered phase in the plasma membrane of Arabidopsis root cells labelled by the PA probe was 500-550 nm, while the emission spectrum of the liquid-disordered phase was 580-700 nm. Treatment of wild-type plants with the sterol extraction agent MβCD resulted in a decrease in plasma membrane order. In the double mutant smt2/smt3 lacking the key methyltransferase in sterol synthesis, the plasma membrane order was consistent with that of the wild-type plants after treatment with MβCD. In the smt2/smt3 mutant, the plasma membrane order of the root hair cells was lower than that of the plasma membrane order of wild-type root hair cell, indicating that sterols, as key components of membrane microdomains, play an important role in regulating the order of the plasma membrane. This study provides a straightforward and rapid detection method for monitoring the dynamic characteristics of living plant cell membrane and changes in membrane microdomains.