The pH value of TE buffer, proteinase K addition, absorption time and temperature were optimized during the absorption process. Under optimized conditions, a trace amount of genomic DNA of one or more types of pathogens can be adsorbed directly by amino magnetic nanoparticles in powdered infant formula (PIF). Combined with specific amplification of Cronobacter sakazakii by PCR identification, the detection sensitivity can reach 102 CFU/mL. Besides, the control test based on this method showed a stronger specificity of common food borne pathogens and the main species of Cronobacter spp.. And the interference test proved that DNA of other interfering strains did not affect the detection of C. sakazakii. This method avoided the use of antibodies, solved the problems of difficult preparation and high cost of antibody, overcame the long pre-enrichment incubation time of traditional method and facilitated the rapid detection of C. sakazakii in PIF.