Aims: The purpose of this study was to develop a general method for the facile development of a new DNA biosensor which utilizes streptavidin-displayed spores as a molecular machinery. Methods and Results: Fluorescence spectroscopy was used as a monitoring tool for the streptavidin displayed on the surface of Bacillus thuringiensis spores and as a diagnosis method for DNA detection. As a proof-of-concept, four pathogenic bacteria including Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumonia were used for the detection of pathogenic species. In addition, a set of mutant variants of Wilson's disease were also used for the detection of single nucleotide polymorphism (SNP) in this system. Conclusions: This strategy, utilizing streptavidin-displayed spores, is capable of capturing DNA targets for the detection of pathogenic bacteria and for mutation analysis in Wilson's disease. Significance and Impact of the Study: This approach could be useful as a simple platform for developing sensitive spore-based biosensors for any desired DNA targets in diagnostic applications.