A highly sensitive method based on mass-barcoded gold nanoparticles (AuNPs) and immunomagnetic separation has been developed for multiplex allergy diagnosis by MALDI mass spectrometry in a component-resolved manner. Different analytical probes were prepared by coating AuNPs with individual allergenic proteins and mass barcode, represented by polyethylene glycol molecules of various chain lengths. Magnetic beads (MBs) functionalized with antihuman IgE antibodies (Abs) were used as immunomagnetic capture probes. IgE Abs were extracted from a patient’s blood serum by the formation of a sandwich structure between the AuNPs and MBs. Multiple specific IgE Abs were simultaneously identified by mass spectrometry detection of the mass barcodes, providing an efficient component-resolved allergy diagnosis. Because of the signal amplification provided by the mass barcodes, the developed diagnosis method is very sensitive, with a limit of detection down to picograms per milliliter level for specific IgE Abs. The method can be potentially useful when the sample amount is highly limited and a multiplex diagnostic procedure is required.