A novel label-free electrochemical immunosensor was developed for sensitive and reliable detection of PthA effector protein, for citrus canker diagnosis. In this approach, Prussian blue (PB), as an electron transfer mediator, was electrodeposited onto the carbon nanotube-ionic liquid (BCNT-IL) modified glassy carbon electrode. In the next step, gold nanoparticles (GNP) were electrodeposited onto the as-prepared film for biomolecules anchoring. The morphology of modified electrode surface was studied by means of field emission scanning electron microscope (FE-SEM). Moreover, the role of each component in the modified electrode was further investigated by cyclic voltammetry (CV). The results indicated that the proposed modification strategy effectively enhanced the stability and electroactivity of PB. After the immobilization of anti-PthA antibody, the immunosensor was used for determination of different concentrations of PthA antigen under the optimal conditions, using Fast Fourier transform square wave voltammetry (FFT-SWV) technique. The dynamic linear range of this electrochemical immunosensor for detecting PthA protein was from 0.03 to 100 nM, with a detection limit of 0.01 nM. The prepared immunosensor displayed high selectivity, long term stability, good repeatability and great potential for application in real sample analysis.