Humic substances are refractory organic compounds that are relatively low in biological activity but known to stimulate phytoplankton growth in estuarine and marine environments. The effect of humic substances on phytoplankton growth was investigated in the seawater zone of the Yeongsan River estuary where an episodic anthropogenic freshwater is discharged, affecting its water properties directly. Water samples and data of water properties were collected at three stations (Sts. A–C) along the channel of the seawater zone in February, May, August and November, 2009. The collected water samples were incubated after humic acids (HA) were added in mesocosm experiments. Phytoplankton (chlorophyll-a) were fractionated into net- (〉 20 μm) and nano-size (〈 20 μm) to examine the response of phytoplankton according to size. Their response to HA treatment was assessed by repeated measures analysis of variance (RMANOVA). The experiments showed that phytoplankton biomass (chlorophyll-a) significantly increased after HA were added at the stations near the sea dike. Especially, nano-sized chlorophyll-a concentrations increased significantly throughout the seasons. This indicates that understanding the behavior of refractory organic matters such as humic substances is required to better manage altered estuarine ecosystems including the Yeongsan River estuary which are affected by episodic discharge of freshwater from sea dikes.