The invention discloses a magnetic bead preparing method and application, and belongs to the technical field of magnetic material preparing. The preparing method comprises the steps that firstly, magnetic nanoparticles with the surface functionalization are prepared, then, at least two magnetic nanoparticles with the surface functionalization are connected through a cross-linking agent, and a magnetic bead with the magnetic content of 70 %-98 % is prepared. The problems that the magnetic content of an existing magnetic bead is low, to achieve fast separation, the size of the magnetic bead is greatly increased, and consequently the suspension stability is reduced are solved. In addition, for the captured component biological reaction, particularly for the immunoreaction, the steric hindrance caused by the small magnetic bead is obviously reduced, the capturing capability and the analyzing and detecting sensitivity are improved, the magnetic bead can be better applied to the biological medicine separation and analysis, and the magnetic bead is particularly suitable for separation of cells, bacteria, viruses, DNA/RNA, protein and the like in biological body fluid or reaction fluid and the clinical biochemistry fast automatic detection based on chemiluminiscence electrochemical luminescence and fluorescence.