A label-free electrochemical immunosensor, based on a gold nanoparticles (Au NPs)/silicon template/methylene blue (MB)/chitosan (CHIT) nanocomposite-modified electrode, was fabricated for the ultrasensitive detection of microcystin-LR (MC-LR). The nanocomposite film showed high binding affinity to the antibodies of MC-LR because gold nanoparticles have large surface area and good biocompatibility which can immobilize large amount of antibody through ionic interactions and other interactions between AuNPs and mercapto or primary amine groups of antibodies with high stability and bioactivity. MB was used as redox indicator due to its good electrochemical behavior in conductive substrate. This hybrid membrane was evaluated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) to determine its electrochemical properties in immunosensor application. A decrease in DPV responses was observed with increasing concentrations of MC-LR in standard and real samples due to the formation of immuno-complexes between MC-LR and anti-MC-LR antibodies which hindered the electron charge transfer at the electrode-electrolyte interface. At optimal conditions, this immunosensor could detect MC-LR in a linear range from 0.5 ng/mL to 25 microg/mL with a low detection limit of 0.1 ng/mL at 3 sigma. Moreover, the prepared immunosensor was applied for the analysis of MC-LR in water samples with satisfactory results. The proposed method showed high selectivity, acceptable reproducibility, stability and reliability.