Nowadays, hydrogels have been attracted a lot of interest due to their immense potential in different fields such as biomedicine and biotechnology. Biodegradable and biocompatible pH-sensitive chitosan-grafted polyacrylic acid (CTS-g-PAA) hydrogel was synthesized by grafting an acrylic acid monomer onto chitosan at the presence of methylene bisacrylamide as a cross-linking agent and ammonium persulphate as an initiator. FT-IR spectroscopy and scanning electron microscopy (SEM) were used to analyze the properties of the obtained hydrogel. The synthesized hydrogel is suitable for the delivery of many hydrophilic drugs or species. Using a multi-walled carbon nanotube modified-glassy carbon electrode (CNT-GCE), the loading and release conditions of Nile Blue (NB) as an electroactive compound were evaluated utilizing the differential pulse voltammetry (DPV) and cyclic voltammetry (CV). The effect of various parameters on the electrochemical signal of NB was investigated, and the optimal conditions for the efficient performance of hydrogel to delivery of NB were obtained. The electrocatalytic current values show linear dependence to NB concentration in the range of 0.098 – 0.971 μM while the detection limit of this electrochemical platform was 12.3 nM. The unique proposed hydrogel with the electroactive NB has a broad range of possible applications in biosensors for signal enhancement and bioanalysis. Supplementary Information The online version contains supplementary material available at 10.1007/s10854-021-06939-7.