Developing sensors for pathogens using real microorganisms for initial testing of the device can introduce additional variables and potential experimental hazards. The artificial microorganism “Bugbead” could be used as a simulant to evaluate the device and for methods development. Bugbead consists of a microsphere coated with proteins to represent the epitopes on a real microorganism. The advantages of the Bugbead include those of being non-pathogenic, similar in bead size to a real microorganism, and having a controllable epitope density. A well-characterized Bugbead eases the study of the device and can be prepared, stored and used safely. Bead-based sandwich immunoassay was done using, as a model system, biotinylated sheep–anti-mouse IgG as a primary antibody immobilized on streptavidin coated 2.8 μm magnetic beads. Neutravidin-dendrimer coated 0.39 μm nonmagnetic Bugbeads were labeled with biotinylated mouse and guinea pig IgGs. Alkaline phosphatase conjugated donkey–anti-guinea pig IgG was the secondary antibody. p-Aminophenyl phosphate was the phosphatase substrate and the p-aminophenol product was detected by rotating disk electrode-amperometry. The minimum detected amount was 360 Bugbeads. The capturing event between magnetic capture beads and Bugbeads was observed by optical microscopy. In simple tests of the concept, an extract containing serotype antigens of E. coli O157:H7 were coupled to Bugbeads and examined in two modified commercial test kits for this pathogen. In both cases positive results was obtained, indicating that the pathogen antigens had been successfully transferred to the microbead and were capable of simulating the assay response to the pathogen itself. These experimental results demonstrate that the Bugbead could be used in the initial steps of developing microbial sensors.