Planktonic protists, including both autotroph and heterotroph, have been recognized as a major contributor to primary production and consumers of bacteria, archaea and picophytoplankton. However, the understanding of protistan diversity is typically impeded by the large amount of metazoans when employing universal primers to environmental samples. In this study, we developed 3 blocking primers to inhibit the amplification of metazoan DNA in PCR. First, we optimized the design and concentration of blocking primers by using 2 metazoans and 3 protists in quantitative PCR. No significant difference (q > 0.05) was found in protistan community structure at phylum, family or OTU levels and genetic diversity between samples amplified with and without adding blocking primers, indicating that the blocking primer does not alter the composition of protistan community. An application to samples with a high abundance of metazoans showed that the blocking primer can reduce 42.1-72.4% of metazoan sequences, resulting the retrieval of a higher protistan richness and diversity. The use of the blocking primer helped to identify protistan community composition from a large size fraction, which is usually dominated by metazoans. Protistan community from the small (0.2-10 mu m) and large (> 10 mu m) size fractions exhibited a low similarity of 36.6% and shared 14.7% OTUs. About 63.8% of the OTUs were unique to the large fraction. Species from groups, such as Lepidodinium, Warnowia, Kareniaceae, Torodiniales, Phaeocystis, Chrysochromulina and Chrysophyceae, were enriched in the large fraction, indicating that they could be largely underestimated in studies that exclude the large-sized cells. Blocking primers are a promising tool to increase the efficiency for the characterization of protistan diversity in aquatic planktonic ecosystems.