Alternative electrode materials suitable to prepare novel working electrode applicable in detecting biopolymers such as nucleic acids, proteins or glycoproteins, represent a significant contribution to bio‐electroanalysis. Herein, electrodes made of vapor‐deposited thin gold films (vAuE) were used as an alternative substrate for the electrodeposition of silver amalgam particles (AgAPs), next to indium tin oxide and pyrolytic graphite, which are already used. The conditions and parameters of double pulse chronoamperometry were optimized for the most‐sensitive voltammetric detection of 4‐nitrophenol (4‐NP). The resulting electrodes were characterized by scanning electron microscope with energy dispersive X‐ray spectroscopy. While 4‐NP could not be detected by bare nonactivated vAuEs at all, their electrochemical activation offered a limit of detection (LoD) of 25 and 5 μmol.l−1 by means of CV and DPV, respectively. AgAP electrodeposited on vAuE, offered 2.5‐times lower LoDs 10 μmol.l−1 by CV and comparable LoD 5 μmol.l−1 by DPV. Advantageously, AgAPs could be repeatedly deposited on and anodically dissolved from the vAuE with a relative standard deviation 13 % of the ten‐times repeated DPV signal of 4‐NP (100 μmol.l−1). In comparison to vAuE, the vAuE‐AgAP offered about 400 mV broader potential window, which allowed detection of single strand DNA fragment labeled by osmium tetroxide−bipyridine complex down to 2 ng.μl−1 by means of DPV.

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