A novel immunosensor using eggshell membrane for determining the human immunoglobulin M (HIgM) in serum was developed. The immunosensor was fabricated by immobilizing goat anti-human IgM antibody on the eggshell membrane with glutaraldehyde. Based on the immunoreactions of goat anti-human IgM (primary antibody), HIgM (target antigen) and the goat anti-human IgM (secondary antibody), the sandwich complex were formed on the eggshell membrane and fluorescein isothiocyanate (FITC) labeling secondary antibody could be employed to detect the target antigen. Under the optimized conditions, the linear range for determining HIgM is 5-60 ng mL(-1) and the detection limit is 4.3 ng mL(-1), which are comparable with the results obtained by general immunonephelometric method. Meanwhile, this proposed sensor also exhibited remarkable storage stability, permeability and highly biocompatibility. The effects of temperature and pH value on eggshell membrane were investigated. Therefore, the proposed immunosensor, by using eggshell membrane as immobilization platform of antibody, offers an excellent fluorescence response to HIgM. The immunosesor provided a new alternative to determine antigens and other bioactive molecules.