Bacterial infection is a significant cause of morbidity and mortality to humans worldwide. Thus, a method for nonspecific, sensitive, and rapid enrichment of such bacteria is essential for bacteria detection and treatment. This study demonstrates a self-made core-shell Fe3O4@Polydopamine@Polyethyleneimine magnetic beads (Fe3O4@PDA@PEI MBs) with a high density positive charge-based magnetic separation scheme for the broad-spectrum rapid enrichment of microorganisms in the liquid phase. MBs with a high-density positive charge have a strong electrostatic attraction to most microorganisms in nature. Our scheme is as follows: (1) wrapping dopamine (DA) on the iron oxide through self-polymerization and wrapping PEI on the outermost shell layer in a mode of crosslinking with the PDA; (2) subsequently, the Fe3O4@PDA@PEI MBs were used to concentrate microorganisms from the sample solution; (3) performing magnetic separation and calculating the adsorption efficiency. The as-prepared Fe3O4@PDA@PEI MBs composite was carefully characterized by zeta potential analysis, Value stream-mapping (VSM), transmission electron microscopy (TEM), and Fourier transforms infrared spectrometry (FT-IR). In this study, both gram-positive and gram-negative bacteria could be captured in three minutes through electrostatic interaction. Furthermore, the adsorption efficiency on gram-negative (>98%) is higher than that on gram-positive (>95%), allowing for a simple, rapid assay to enrich organisms in resource-limited settings.