A novel method based on Cu-chelated magnetic beads (Cu-Magbeads) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the rapid detection of peptide toxins in serum. The peptides in the serum were efficiently adsorbed by the Cu-Magbeads, eluted with methanol solution, and assayed by MALDI-TOF-MS. Specific peptides were identified according to their characteristic mass-to-charge ratio values. Conotoxin SO(3), a synthesized peptide, was used as a model to evaluate the method. Conotoxin SO(3) was detected in human serum, as well as bovine and murine serum, with a detection sensitivity in the low femtomole range. The assay was performed within 40 min, without the need for a specific antibody or an expensive reagent. It shows potential for future use in clinical and emergency rescue practice because of its simplicity, high speed, and high sensitivity.