In recent years, brown tide is a new type of marine ecological disaster in China, caused by picoalgae. Due to the small size of individuals, picoalgae is difficult to be identified by the traditionally morphological taxonomy methods. At present, studies on its outbreak mechanism, ecological distribution, monitoring and prevention methods are less. In this study, we designed a pair of primers (V4F/R) in 18S rDNA for eukaryotic microalgae indification and a biomolecular identification method for brown tide was established. The results of primers sensitivity and specificity evaluation showed that the primers V4 (F/R) were higher than other primers in amplification specificity, species diversity excavation and dominant species identification of eukaryotic picoalgae. A total of 136 species of eukaryotic microalgae and picoalgae were detected in Liaodong bay by this method, including autotrophic type accounting for 60%, mixotrophic type for 30% and heterotrophic type for 10%. Among them, 56 species have not been reported in Chinese waters, and another 30 species have not been reported in Liaodong Bay, but reported in other Chinese waters. This study is of great significance for the enrichment of phytoplankton in Chinese waters and the background database of exotic marine microalgae.