With the advancement of gene sequencing technology, researchers have been able to uncover microalgae (e.g., picocyanobacteria) that were previously unknown in the environment. Despite this, previous publications shed limited light on the comprehensive taxonomic analysis of picocyanobacteria in China. This study focused on the isolation and purification of Cyanobium gracile HNU001 from a waterworks, which was the first study reporting this species in detail. The morphology, ultrastructure, and molecular characteristics of the organism were investigated. Morphological observations showed an ellipsoidal or elongated shape of the algal cells that divided symmetrically into shorter cells. Ultrastructural analysis indicated that cell division through dimitosis and the arrangement of thylakoid membranes were parallel to the cell membrane. Analysis of the 16S rRNA gene sequences demonstrated a high similarity of over 99.9% between this strain and Cyanobium, placing it within a distinct molecular cluster. Comparative analysis of the D1-D1′ and Box-B helical structures of Cyanobium gracile HNU001 with Cyanobium gracile PCC 6307 showed high consistency. Based on these findings, the species was formally named Cyanobium gracile, i.e., the first taxonomic characterization in China.