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An amperometric biosensor using a substrate recycling principle was realized for the detection of low adrenaline concentrations (1 nM) by measurements in phosphate buffer and Ringer’s solution at pH 6.5 and pH 7.4, respectively. In proof-of-concept experiments, a Boolean logic-gate principle has been applied to develop a digital adrenaline biosensor based on an enzyme AND logic gate. The obtained results demonstrate that the developed digital biosensor is capable for a rapid qualitative determination of the presence/absence of adrenaline in a YES/NO statement. Such digital biosensor could be used in clinical diagnostics for the control of a correct insertion of a catheter in the adrenal veins during adrenal venous-sampling procedure.