Abstract: This study attempted to establish a new rapid assay for adenosine combined adenosine deaminase(ADA) with indophenol method. Based on the single factor tests,the optimal reaction conditions were explored by response surface methodology with four variables of sodium hydroxide,salicylic acid,sodium nitroferricyanide and sodium hypochlorite. And a verification experiment using the optimum composition( sodium hydroxide25.85g/L,salicylic acid 68.05g/L,sodium nitroferricyanide 2.19g/L,sodium hypochlorite 40.9m L/L) was performed.A adenosine standard curve was established under the optimum reaction conditions in range of 0.01~10mmol/L.The recovery of adenosine by ADA assay were between 95.5%~103.8% and RSD were between 0.9%~3.3%.This assay showed high consistency with HPLC method when determine the concentration of adanosine in fermentaton broth. Then the reliability of the ADA assay was further supported by spike and recovery test. The results showed that the method could rapidly and high-throughput determine the adenosine concentration in fermentation broth.