The plasma membrane is a dynamic biointerface, yet its long-term visualization under physiological culture conditions remains challenging. Conventional plasma membrane probes often suffer from progressive intracellular internalization and nonspecific serum-associated fluorescence. Herein, we report PM580, a serum-resistant fluorogenic probe that enables long-term, wash-free, and surface-confined plasma membrane imaging in live cells. PM580 remains weakly emissive as quenched supramolecular assemblies in aqueous media but becomes strongly fluorescent upon redistribution into lipid bilayers. Structure-property analysis and molecular modeling suggest that the carboxylic acid-containing acceptor may serve as an interfacial positioning element that favors a membrane-inserted orientation, whereas excessive alkyl-chain extension promotes intracellular accumulation and compromises surface selectivity. This balanced architecture enables strong plasma membrane activation with minimal responses to serum albumin or mucin, allowing direct imaging in complete medium and mucin-rich intestinal epithelial monolayers. PM580 further enabled real-time visualization of junctional disruption, membrane tethering, and cell-cell dissociation induced by P. gingivalis conditioned medium. These findings identify a favorable structural balance that supports serum-compatible and long-term plasma membrane imaging.