We demonstrate that alcohol oxidase, in combination with paraformaldehyde and catalase, enables rapid and complete oxygen removal from microliter-volume electrochemical biosensors. Like many oxidases, alcohol oxidase is highly specific for its reducing substrate, namely short chain primary alcohols and paraformaldehyde. Hence AOx is suitable as biocatalyst for O2 removal in biosensing of any analyte other than its specific substrates. Unlike most oxidase, alcohol oxidase is highly specific for its reaction with O2 serving as oxidizing substrate. This distinctive feature is critical for preventing AOx from donating electrons to the classical electron mediators, such as ferrocendimethanol and Os-complex modified polymers, used in electrochemical biosensing. Therefore, the AOx-based oxygen scavenging system does not interfere with the electron transfer pathway responsible for signal detection.