In this study, nanophytoplankton and picophytoplankton diversity in seawater samples was analyzed using a high-throughput sequencing platform and a series of bioinformatics tools, based on the V4 and V9 region of 18S rDNA as the target gene. High-throughput sequencing, which is considered as one of the most important tools in genomics research, is widely applied in the field of marine nanophytoplankton and picophytoplankton diversity studies. We successfully obtained a pair of nanophytoplankton and picophytoplankton PCR primers V4(F/R)by analyzing the nucleic acid database and using a series of bioinformatics tools. Two pairs of universal primers were also selected for comparative analysis, which amplified variable region V4 and V9 of the small subunit nuclear ribosomal DNA (SSU nrDNA).The sensitivity and specificity of PCR primers V4(F/R), V9(F/R), and C4(F/R) were also evaluated and compared using online bioinformatics software. The results showed that the amplification specificity of primer pair V4(F/R) was better than that of V9(F/R) and C4(F/R) in eukaryotic algae. High-throughput sequencing results showed that 68834 raw tags were amplified by the primers, 99% of which were effective tags. Sequences of more than 94% of the effective tags were identified by Ribosomal Database ProjectClassifier, among which 308 operational taxonomic units (OTUs) of one sample were used for further analysis. The average numbers of nanophytoplankton and picophytoplankton OTUs amplified by V4(F/R), V9(F/R), and C4(F/R),were 78, 42, 58,respectively. The primer pair V4(F/R) was found to have higher sensitivity and specificity for amplifying nanophytoplankton and picophytoplankton, including Micromonas pusilla,Ostreococcus tauri,Pycnococcus provasolii,Aureococcus anophagefferens,and Heterosigma akashiwo. The V4 region from the environmental eukaryotic 18S rDNA gene could be suitable for high-throughput sequencing technology, and it was also a good target gene formarine nanophytoplankton and picophytoplankton identification. This study demonstrates the use of a simple, rapid, high sensitivity, and low-cost technology to explore marine nanophytoplankton and picophytoplankton diversity. Moreover, it also provides a reference for the early warning and control of brown tide disasters.