Adenosine is one of the nucleoside which plays an important role in signal transduction and neuromodulation. This work proposes a simple electrochemical assay, comprising two enzymes and rhodium complex based electron transfer mediator, for the detection of adenosine. Sequential reaction of adenosine deaminase and l-glutamic dehydrogenase and the supporting cycle between beta-NADH and mediator enable quantitative analysis of adenosine. Role of electron transfer mediator is the conveyance of proton from electrode to beta-NAD(+) for regeneration of beta-NADH. The electrochemical characteristics of electron transfer mediator were also studied. Real-time adenosine detection was carried out using this multiple enzyme based chronoamperometric assay. The analysis results show a low limit of detection (140 mu M) and good correspondence between current signal and the adenosine concentration (R-2 = 0.997).