A procedure to the trace amount of biological agents in water, based on fluorescence correlation spectroscopy and confocal microscopy, is explored. A special flow system is developed to increase the probability of detecting Escherichia coli (E. coli), and confocal microscopy is used to increase the sensitivity of the detection system. It is observed that concentrations higher than 1.5×105 E. coli per milliliter (2.5×10-16 M) are detectable with the present setup. This concentration corresponds to about 1.0 nM level of Rhodamine 6-G dyes. For detection of lower concentrations, further improvements need to be implemented to increase signal/background ratio. A detailed analysis of the optical system is presented and further improvements for the procedure are discussed.