In developing a useful tool to detect parasitic dynamics in an estuarine ecosystem, a denaturing high-performance liquid chromatography (DHPLC) assay was optimized by cloning plasmid DNA from the grass shrimp Palaemonetes pugio, and its two parasites, the trematode Microphallus turgidus and bopyrid isopod Probopyrus pandalicola. The optimal separation condition was an oven temperature of $57.9^{circ}C$ and 62-68% of buffer B gradient at a flow rate of 0.45 mL/min. A peptide nucleic acid blocking probe was designed to clamp the amplification of the host gene, which increased the amplification efficiency of genes with low copy numbers. Using the DHPLC assay with wild-type genomic, the assay could detect GC Gram positive bacteria and the bopyrid isopod (P. pandalicola). Therefore, the DHPLC assay is an effective tool for surveying parasitic dynamics in an estuarine ecosystem.