Bacterial spores commonly co-exist with vegetative cells, presenting challenges in spore separation and detection. The separation of spores is a crucial process for laboratory research and the detection of spore mechanisms. This study introduced a novel method that leverages the high binding affinity of vancomycin (Van) and ampicillin sodium (Amp) to vegetative cells, integrated with magnetic separation technology, to selectively collect spores from complex environments by eliminating vegetative cells. First, Van/Amp-modified magnetic Fe3O4 nanoparticles (Fe3O4 NPs) were synthesized and characterized. Subsequently, these NPs bound vegetative cells, forming magnetic conjugates that could be efficiently removed using a magnetic field. Concurrently, spores were collected with an efficiency exceeding 95%, completing the entire process within 30 min and achieving a spore separation efficiency of up to 105 CFU/mL. This method was successfully applied to actual samples, including tap water and milk. The state of the collected spores was confirmed using Raman spectroscopy and microscopic techniques, verifying that their characteristics matched those of typical spores. The proposed novel method for rapid spore separation, leveraging the "remove bacterial effect" facilitated by Van/Amp-Fe3O4 NPs, showed outstanding spore collection capabilities while preserving the excellent physiological state of spores.