Cell surface proteins (CSPs) are valuable targets for therapeutic agents, but achieving highly selective CSP enrichment in cellular physiology remains a technical challenge. To address this challenge, we propose a newly developed sulfo-pyridinium-ester (SPE) cross-linking probe, followed by two-step imaging and enrichment. The SPE probe shows higher efficiency in labelling proteins at the level of cell lysates than similar NHS esters and demonstrates specificity for Lys in competitive experiments. More importantly, this probe can selectively label cell membranes in cell imaging, while only negligible labelling of the intracellular compartment was detected. Moreover, we successfully performed this strategy to mainly label the cell surface proteins in MCF-7 live cells. Finally, we used our probe to label CSPs under insulin treatment, revealing several cell surface targets for key functional biomarkers and the pathogenesis of insulin-stimulated MCF-7 breast cancer cells. The above results show that the SPE method provides a promising tool for selectively labelling cell surface proteins and monitoring transient cell surface events.