Phytoplankton exopolymeric substances (EPS) contribute significantly to the dissolved organic carbon (DOC) pool in the ocean, playing crucial roles in the surface ocean carbon cycle. Recent studies have demonstrated that ~10% of marine DOC can self-assemble as microgels through electrostatic Ca bonds providing hotspots of enriched microbial substrate. However, the question whether EPS can self-assemble and the formation mechanisms for EPS microgels have not been examined. Here were report that EPS from three representative phytoplankton species, Synechococcus, Emiliania huxleyi, and Skeletonema costatum can spontaneously self assemble in artificial seawater (ASW), forming microscopic gels of ~ 3 - 4 μm in diameter. Different from the marine DOC polymers assembly, these EPS samples can self-assemble in Ca2+ -free ASW. Further experiments from fluorescence enhancement and chemical composition analysis confirmed the existence of fair amounts of hydrophobic domains in these EPS samples. These results suggest that hydrophobic interactions play a key role in the assembly of EPS from these three species of marine phytoplankton.