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The purpose of this paper is to study the preparation of magnetic nanoparticles (MNPs)by solvothermal method with PEG-4000 as surfactant, the surface modification of the MNPs byseveral steps, rapidly detection method of pseudomonas aeruginosa based on magnetic separationand chemiluminescence. The results shown that we prepared a kind of MNPs with uniform shapand good dispersed, and the sizes of MNPs can be controlled by adjusted the dosage ofFeCl3·6H2O. SEM, TEM and FTIR showed that the several surface modified method of MNPs isfeasible to gain probe-modified MNPs. The biotin-dUTP-labeled DNA fragments of gyrB genewas amplified by PCR, and the method of pseudomonas aeruginosa detection based on preparingMNPs has been developed in this work.