ObjectiveTo develop a multiplex touchdown PCR for simultaneous detection of extendedspectrum βlactamases (ESBLs)producing Enterobacteriaceae and methicillinresistant Staphylococcus aureus (MRSA).MethodsBlood culture positive specimens from 102 hospitalized patients were collected between March 2013 and  September 2014, four pairs of specific primers were designed based on SHV,TEM, and OXA genes of ESBLsproducing Enterobacteriaceae and MecA gene of MRSA,  drugresistant genes were amplified with single touchdown PCR and multiplex touchdown PCR, the results were compared with KirbyBauer disk diffusion method.ResultsEach single PCR amplified a specific band， four drugresistant genes were also detected by multiplex touchdown PCR；the lower detection limits of multiplex touchdown PCR for DNA of  MecA,SHV, TEM, and  OXA were 4.37 ng, 2.19 ng, 4.53 ng, and 3.59 ng, respectively. Compared with KirbyBauer disk diffusion method, the overall sensitivity and  specificity of multiplex touchdown PCR were 100.00% and 88.24% respectively, for ESBLs were 100.00％ and  87.23％ respectively，for  MRSA were both 100.00％.ConclusionA higher sensitivity and specificity multiple touchdown PCR assay has been developed, and it can be used in the rapid diagnosis and epidemiology investigation of  bloodstream infection caused by ESBLsproducing Enterobacteriaceae and  MRSA, and is helpful for guiding antimicrobial use in clinic.