To develop an antimicrobial activity detection method of enzyme preparations derived from microorganism, the key factors including paper specification, medium, the concentration of microorganisms as well as positive control antibiotics were screened and examined, based on the method recommended by JECFA. MethodsTaking 5.0and 10.0μg/piece of ciprofloxacin as positive control, nine kinds of paper, three kinds of medium and three bacteria concentrations under the same conditions (temperature, incubation time, etc) were checked for their influence on antimicrobial activity. ResultsThe effect of HZT No.2and Whatman 903(2piece superposed) were comparable with the commercialized antimicrobial determining paper. The effect of tryptone soy agar (TSA) domestically produced (defined as No.1and No.2) were comparable with imported TSA. The appropriate dilution of tryptone soy broth to 24h cultures was 10∶1 for 5reference stains, and 20∶1 for Streptococcus pyogenes. The recommended concentration for ciprofloxacin antimicrobial activity test was 5.0μg/piece.ConclusionPaper HZT No.2or Whatman903(2piece superposed) or whatman 2017-013, domestic or imported TSA medium, 10times culture dilution (20times for Streptococcus pyogenes), and 5.0μg/piece ciprofloxacin were recommended in the determination of antimicrobial activity of enzyme preparations derived from microorganism.