In the current study, an electrochemical biosensing signal amplification system was utilized with thionine-chitosan-gold nanoparticles (Chit-GNPs) that absorbed horseradish peroxidase (HRP) and anti-His tagged protein monoclonal antibody derived from Balb/c mice. In addition, transmission electron microscopy (TEM) was used to characterize the nanogold solution and atomic force microscopy (AFM) was used to characterize the sensor assembly. To evaluate the quality of the immunosensor, the amperometric I-t curve method was applied to determine His-IL23 in PBS. The results indicated that the response current exhibited an optimal linear correlation with the His-IL23 concentration that ranged from 0.01 to 103 ng/ml. The lowest detection limit was noted at 3.3 pg/ml (S/N = 3). The linear equation was deduced as follows: △I = 0.02lgC + 0.037 (R2 = 0.9628). Moreover, it was validated with high sensitivity, reproducibility and rapid response. Apparently, the immunosensor may be a very useful tool for the detection and quantification of His-tagged proteins. In addition, the signal amplification system can be used for the preparation of other immunosensors and to assist in bioassays.