Electrodeposition of silver amalgam particles (AgAPs) on various substrates offers perspective tool in development of novel electrochemical detection system applicable even in direct bioelectrochemistry of nucleic acids or proteins. Herein, a double pulse chronoamperometric deposition of AgAPs on in-house fabricated screen-printed silver electrodes (SPAgE) has been optimized using voltammetric signal of model electrochemically reducible organic nitro-compound, 4-nitrophenol, and scanning electron microscopy with energy dispersive X-ray spectrometer. This compact sensor including graphite counter and Ag|AgCl pseudo-reference electrode was design for highly effective analysis of electrochemically reducible compounds in 96-well plate with about 150-µl sample volume per well. The SPAgE-AgAP offers detection of 4-NP down to 5 µmol.l⁻¹ using cyclic voltammetry in acetate buffer pH 5.0. Advantageously, differential pulse voltammetry at SPAgE-AgAP allows highly sensitive detection system of unnatural nucleosides dTPT3 and d5SICS, which successfully expanded genetic alphabet of recently studied semi-synthetic organism, with limits of detection 0.1 pg.µl⁻¹ in 0.05 mol.l⁻¹ hydrochloric acid. Moreover, these artificial nucleosides are detectable in the mixture with natural nucleosides up to weight ratio 1 : 15 000. SPAgE-AgAP may be potentially utilized in simple, fast and sensitive electrochemical detection of organic nitro compounds and free or in DNA harbored dTPT3 and/or d5SICS, what may contribute in successful research and development of semi-synthetic organism perspective in chemical and synthetic biology.