• A novel ECL system consisting of nalidixic acid (NA) and K 2 S 2 O 8 was investigated. • 4-Pyridinecarbohydrazide as a co-reactant accelerator that significantly amplifies the ECL signal of the NA system. • A tetraethylenepentamine detection strategy based on ECL technology was developed. This work presents a method for the establishment of a highly sensitive and selective nalidixic acid and S 2 O 8 2- (NA/S 2 O 8 2- ) electrochemiluminescence (ECL) system using isonicotinic (INH) as a co-reaction accelerator. After optimizing the experimental conditions including scan rate, pH value of buffer solution, concentration, etc., the chemiluminescence mechanism of the system was studied in detail using fluorescence spectroscopy, ECL spectroscopy and cyclic voltammetry (CV). The results show that INH is an efficient co-reaction accelerator, which interacts with the co-reactant S 2 O 8 2- but not the luminophore NA to promote the production of more oxidant SO 4 ·– , which then further reacts with NA to generate excited state species NA * with strong ECL signal, indicating that co-reaction accelerators can provide a simple, efficient, and low-cost method for signal amplification. Based on the great ECL enhancement of INH for the NA/S 2 O 8 2- system, under the optimal experimental conditions, highly sensitive and selective electrochemiluminescence detection of tetraethylenepentamine (TEPA) was achieved.