A sandwich electrogenerated chemiluminescence (ECL) biosensor was fabricated based on concanavalin A (Con A)-integrating gold-nanoparticle-modified Ru(bpy)3(2+)-doped silica nanoprobe (Au-RuSiO2 NPs) for in situ and dynamically evaluating cell surface N-glycan expression. Owing to the specific recognition of Con A with mannose and the core trimannoside fragment of N-glycan and the effective ECL amplification of Au-RuSiO2 NPs, the as-proposed biosensor exhibited excellent analytical performance toward the cytosensing of K562 cells with a wide detection linear range from 1.0 × 10(3) to 1.0 × 10(7) cells mL(-1) and a detection limit of 600 cells mL(-1). More importantly, the strategy was successfully applied to evaluate cell surface N-glycan expression under different external stimuli of inhibitors and enzyme. This biosensor is endowed with feasibility and reliability of generating sensitive insight into the majority of N-glycan expression on the cell surface. Furthermore, the biosensor was employed to dynamically profile cell surface N-glycan expression at different phases of cell growth in vitro. This biosensor is promising in studying and elucidating the N-glycan function in biological and physiological processes.