El Rayah El Tawfiky is one of the main sources of water supply for 3 governments in the Nile delta, Egypt. The investigation was carried out on the relationship between some physico-chemical parameters and phytoplankton biomass of El Rayah El Tawfiky in the Nile delta, Egypt. Water and phytoplankton samples were collected seasonally from five stations along El Rayah El Tawfiky (from Kafr Moeys in Banha city to Aga in El-Mansoura city) for one year (from October 2018 to September 2019). The highest water temperature was recorded in the spring season, whereas the lowest degree was recorded in the winter season. The pH values of water in El Rayah El Tawfiky at all stations ranged from slightly alkaline to alkaline. There was a wide range in the degree of turbidity during the seasons. There was a relative increase in the nitrite, nitrate, and Orthophosphate contents during the winter. The detected phytoplankton community consisted of 132 species of five systematic algal divisions (Chlorophyta, Bacillariophyta, Cyanophyta, Pyrrophyta, and Euglenophyta). Qualitatively, Chlorophyta was the dominant division followed by Bacillariophyta, Cyanophyta, Euglenophyta, and Pyrrophyta. On the other hand, the quantitative study showed that Bacillariophyta was the most dominant division, followed by Chlorophyta, Pyrrophyta, and Euglenophyta. The highest biomass was recorded during the winter, this may be related to the presence of excess nutrients especially orthophosphate, nitrate, and nitrite during this season. Additionally, the lowest biomass was recorded during the spring; this may be attributed to the presence of small amounts of nutrients and an increase in water turbidity.  Moreover, the current study revealed that there were different relationships between total phytoplankton biomass and physico-chemical parameters. Phytoplankton biomass had a positive relationship with nitrite, and orthophosphate meanwhile a negative relationship with temperature. Moreover, there was a very strong positive relationship between nitrate and Cyanophyta and also a very strong positive correlation between Euglenophyta and ammonia concentrations. Phytoplankton biomass is regarded as an easy and useful indicator means to assess the quality of freshwater ecosystems.