Using bioconjugated dye-doped silica nanoparticles (NPs), we have developed a bioassay for the accurate determination of a single bacterial cell within 20 minutes without any signal amplification or sample enrichment. The antibody-conjugated NPs can specifically and quantitatively detect bacteria, such as Escherichia coli O157:H7 from beef through antibody-antigen recognition. Dye-doped silica NPs have also been successfully used for DNA detection at sub-fentomolar concentrations. Our results demonstrate the potential of dye-doped silica NPs for broad applications in practical biotechnological and medical applications in various biodetection systems. The ultimate goal of integrating bionanotechnology into complex biological systems will emerge as a revolutionary tool for ultrasensitive detection of disease markers and infectious agents.