Most water sources are full of microscopic transparent exopolymer particles (TEP), which are currently regarded as a major initiator of biofilm formation. This study developed and applied an auto-imaging FlowCAM-based method for online observation and quantification of TEP in freshwater. Samples from reservoirs in Taiwan with a wide range of water quality were directly used to develop this methodology. Factors that potentially affect the measurement were tested. The results showed that characteristics of the particles measured instantaneously after staining samples with Alcian blue differed significantly from those measured at steady states, as a result of particle aggregation. Compared to traditional microscopic methods, this proposed method provides a simple, rapid, and less labor-intensive analysis with particle morphological conservation and a large number of particle attributes. By overcoming the limitations from the former, this technique would offer routine monitoring of these transparent particles from various freshwater sources and feed water in membrane filtration, hence facilitating the use of TEP as a critical parameter for biofouling investigation in water treatment. Application of the method for Taiwan reservoirs showed a wide variety of morphological forms of TEP and its abundance, up to 25,000 ppm.