Spread of infecting organisms from patient to patient and hospital surfaces, is usually done by the nurses, doctors and others caring for the patient. The major method of spread is on contaminated hands. β-lactame antibiotics (Penicillin-Cephalosporin)  have selective poisoning and effective to more Bacteria then are very important in cure of diseases. b-lactamase is virulence agent and causes resistance to these antibiotics. According rule of staff hand Bacteria in infection chain, transmission of â–lactamase producing Bacteria in patients, final due to â-lactame antibiotics resistance nosocomial infection in hospital. The subject of this study was survey prevalence of Beta lactamase nano enzyme in isolated bacteria from staff hand of AZZAHRA Hospital in Iran. The present study was performed at one tertiary care hospitals in Isfahan, Iran. During a 24 month period (2007 -2009 ) and 80 of  bacteria isolated from staff hand were studied. Samples collected with finger print method. Standard microbiological methods were performed for detection of bacterial species and for determine â–lactamase production, use Acidimetric method. The collected data was analyzed thorough SPSS version 14 software and Chi-square used for determination of significance of association. The pd” 0.05 was considered significant. According to result, Staphylococcus spp. 28 (35%), Bacillus spp. 48 (60%) Enterobacteriaceae 4 (5%) consist of isolated bacteria. According result of acidometric test from 80 isolated staff hands bacteria 61.9% of strains produce â–lactamase, respectively was in Staphylococcus spp., Bacillus spp. and Enterobacteriaceae 71%, 64.72% and 50%. Establish systems for monitoring antimicrobial resistance in hospitals and the community and link these findings to resistance and disease surveillance data is fundamental to developing treatment guidelines accurately and to assessing the effectiveness of interventions appropriately.