A key challenge for sensor miniaturization is to create electrodes with smaller footprints, while maintaining or increasing sensitivity. In this work, the electroactive surface of gold electrodes was enhanced 30‐fold by wrinkling followed by chronoamperometric (CA) pulsing. Electron microscopy showed increased surface roughness in response to an increased number of CA pulses. The nanoroughened electrodes also showed excellent fouling resistance when submerged in solutions containing bovine serum albumin. The nanoroughened electrodes were used for electrochemical detection of Cu2+ in tap water and of glucose in human blood plasma. In the latter case, the nanoroughened electrodes allowed highly sensitive enzyme‐free sensing of glucose, with responses comparable to those of two commercial enzyme‐based sensors. We anticipate that this methodology to fabricate nanostructured electrodes can accelerate the development of simple, cost‐effective, and high sensitivity electrochemical platforms.