Enzymes are critically involved in maintaining normal cellular physiology through the catalysis of highly specific and tightly regulated chemical reactions. The inhibition or undesired activation of particular enzymatic functions has been associated with the pathogenesis of a number of diseases. Consequently, the aberrant activity of certain enzymes can be regarded as biomarkers for the diagnosis or monitoring of particular diseases. With rapid technological advances in the field of DNA nanotechnology, oligonucleotides have recently emerged as attractive recognition units for monitoring the activity of enzymes compared with organic small molecules or protein antibodies. In this review article, we present an overview of advantages and versatility of the 'label-free' approach for the fabrication of DNA-based sensing platforms using colorimetric and luminescent molecules as signal transducing units and highlight recent examples of label-free strategies that have been employed for monitoring enzyme activity.