In this study, a simple technique was developed for the electrochemical detection of anionic analytes in weakly supported media. This was conducted by the use of electrochemical paper-based analytical devices (ePADs). A sensing platform was modified with nereistoxin and used to determine nitrite as a case study. The electrochemical response was improved due to the accelerated electron transfer between the sensing platform and the nitrite through the electrostatic interaction of the amino group of nereistoxin and the nitrite. The electrocatalytic current of the nitrite in the presence of nereistoxin was enhanced in the weakly supported media. By using nereistoxin as a signal enhancer, 97% of the electrochemical signal was obtained at the low ionic strength of the electrolyte, while less than 35% of this signal was obtained in the absence of nereistoxin. The limit of detection was as low as 20 nM using an ePAD. Generally, the proposed ePAD serves as a promising, efficient and low-cost device for sensing applications in weakly supported media.