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Simple Summary: In order to accurately characterize the composition of the picophytoplankton community by size in the marine environment, we used a size-fractionated filtration plus a high-throughput sequencing molecular method. The results showed that this method can detect the composition of picophytoplankton in different particle size ranges in detail, and it is accurate in assessing the vertical distribution patterns and size of phytoplankton. This method can enrich the study of phytoplankton communities with diameters of less than 2 μm. In this study, flow cytometry (FCM) and size-fractionated filtration, together with high-throughput molecular sequencing methods (SM), were used to investigate picophytoplankton. A particle separation filter and a higher-throughput sequencing method were used to evaluate the composition of a euphotic zone of picophytoplankton—especially picoeukaryotic phytoplankton—in the Western Pacific, and the results of flow cytometry, which is a classic way to detect picophytoplankton, were used as a standard to evaluate the reliability of the results of the SMs. Within a water column of 200 m, six water depths (5, 25, 50, 113 (DCM), 150, and 200 m) were established. In order to further study the particle size spectra of the picophytoplankton, size-fractionated filtration was used to separate water samples from each water depth into three particle size ranges: 0.2–0.6, 0.6–1.2, and 1.2–2 μm. A total of 36 (6 × 3 × 2) samples were obtained through PCR amplification of the 18S rRNA V4 hypervariable region and 16S rRNA, which were biased toward phytoplankton plastids, and then high-throughput sequencing was performed. The estimation of the picophytoplankton diameter relied on forward scattering (FSC) through FCM. The estimation of the vertical distribution and diameter of the picophytoplankton using the SM was consistent with the results with FCM; thus, we believe that the estimation of picophytoplankton composition with the SM has value as a reference, although the size-fractionated filtration seemed to cause some deviations. In addition to Prochlorococcus and Synechococcus, the SM was used to evaluate the composition of picoeukaryotic phytoplankton, which mainly included Prymnesiophycea (Haptophyta) (38.15%), Cryptophyceae (Cryptophyta) (22.36%), Dictyochophyceae (Chrysophyta) (12.22%), and Mamiellophyceae (Chlorophyta) (3.31%). In addition, the SM also detected Dinophyceae (Dinoflagellata) (11.69%) sequences and a small number of Bacillariophyceae (Diatom) (1.64%) sequences, which are generally considered to have large particle sizes. The results of the SM also showed that the picoeukaryotic phytoplankton were not evenly distributed in the euphotic layer, and the vertical distributions of the different picoeukaryotic phytoplankton were different. An analysis of correlations with environmental factors showed that temperature was the main environmental factor controlling the vertical distribution of picophytoplankton. [ABSTRACT FROM AUTHOR]