Abstract A novel immunosensor for Enterobacter sakazakii ( E. sakazakii) based on screen-printed carbon electrode (SPCE) modified by multiwalled carbon nanotube (MWCNT), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF 6) and thionine (Thi) was developed for the first time. The surface morphologies of modified electrodes were characterized by means of atomic force microscope (AFM). The electrochemical performance of the immunosensor was evaluated by cyclic voltammetry. Horseradish peroxidase labeled antibody to E. sakazakii in [BMIM]PF 6 showed high activity and stability. Under optimal conditions, E. sakazakii could be detected in range of 10 3–10 9 CFU/ml with detection limit of 7.7 × 10 1 CFU/ml. The proposed immunosensor exhibited excellent long-time storage stability and had potential use in clinical immunoassay of E. sakazakii.