Ethylenediamine (EDA), a primary biomarker of meat spoilage, has gained considerable attention for its potential uses in the monitoring of meat freshness. Standard analytical approaches have been widely applied for the detection of EDA, meaning that more rapid and convenient detection methods are highly desirable. Here, we introduce an innovative paper-based electrochemical sensor for label-free detection of EDA. To implement a three-electrode system on the surface of paper, silver conductive nano-ink (Ag nano-ink) was drawn by direct pen-on-paper technology. The structure, morphology, and elemental composition of the Ag nano-ink were analyzed using field-emission scanning electron microscopy and energy-dispersive spectroscopy. The presence of the target in the sensing zone changed the electrochemical signal, which was measured using square wave voltammetry. The results demonstrated that the developed sensor detected the target in a linear range from 10 to 1000 µM with a low limit of quantification of 10 µM. Interestingly, the prepared sensing approach demonstrated a strong ability to determine EDA levels in spoiled beef samples. The selectivity of the designed platform was evaluated by assessing the interference of various amino acids. In addition, the prepared paper-based sensor demonstrated excellent stability for four days. We hope that using Ag nano-ink in a novel electrochemical paper-based sensor for recognizing EDA can open a new window for detecting other biogenic amines in the future.