Mixing of freshwater and seawater creates the well-known salinity gradients along the estuaries. In order to investigate how phytoplankton respond to the acute salinity changes, we exposed natural phytoplankton assemblies from the Jiulong River Estuary to differential saline field water while continuously monitoring their photosynthetic performances under both indoor- and outdoor-growth conditions. When the natural cell assemblies from salinity 30 field water were exposed to series low saline field water (salinity 25, 17, 13 and 7.5), the effective Photosystem II quantum yield (ΔF/Fm′) decreased sharply, e.g., to one-fifth of its initials after 5 min exposure to salinity 7.5 field water, and then increased fast during the following 40 min and almost completely recovered after 320 min. During such an exposure process, non-photochemical quenching (NPQ) sharply increased from 0 to 0.85 within 5 min, and then decreased to nearly 0 within the following 70 min. When these cells re-acclimated to salinity 7.5 field water were exposed to series high saline field water (salinity 13, 17, 25 and 30), a similar response pattern was observed, with the decreased ΔF/Fm′ accompanied with increased NPQ, and followed by the recovery-induced increase in ΔF/Fm′ and decrease in NPQ. A similar response pattern as ΔF/Fm′ to the acute osmotic stress was also observed in the photosynthetic carbon fixation capacity according to radiocarbon (14C) incorporation. Our results indicate that estuarine phytoplankton assemblies could rapidly recover from the acute osmotic stress, implying a potential cause for their frequent blooms in coastal-estuarine waters where despite drastically varying salinity, available nutrients are abundant due to the land-derived runoffs or mixing-caused relaxations from sediments.