Glycan-lectin interactions are commonly observed in nature. Analytical methods, which are used to detect lectins that rely on the use of glycan ligand-modified nanoprobes as affinity probes, have been developed. Most of the existing methods are focused on the use of synthetic glycan ligands. Nevertheless, naturally available glycoproteins, such as ovalbumin in chicken egg whites, are good sources for fabricating glycan-immobilized nanoprobes. In this study, we generated functionalized gold nanoparticles (Au NPs) from a one-pot reaction by reacting chicken egg white (cew) proteins with aqueous tetrachloroaurate. The generated Au@cew NPs are mainly encapsulated by ovalbumin, in which the surface is decorated by abundant hybrid mannose and Galβ(1→4)GlcNAc-terminated glycan ligands. Thus, the generated Au@cew NPs containing hybrid mannose and Galβ(1→4)GlcNAc have the capability to selectively bind with their corresponding lectins. Lectins including concanavalin A, banana lectin, and ricin B that have binding moieties toward specific sugars were used as the model samples. Our results showed that the generated AuNPs can be used as multiplex affinity probes for these model lectins. Lectins can be selectively released from the Au@cew NP-lectin conjugates by using specific sugars, such as mannose, glucose, and β-lactose, as the releasing agents to release specific lectins from the conjugates. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used as the tool to characterize the released species from the nanoprobes. The limit of detection of these model lectins using the current approach was low (in nM). The feasibility of using the Au@cew NP-based affinity MALDI-MS to selectively detect specific lectins from complex samples was also demonstrated.