An approach to assess estuarine environmental flow based on phytoplankton preference, including the complex relationships between hydrological modifications and ecosystem biomass, was developed in this study. We initially established a relationship between biomass requirements for primary and higher nutritional level organisms based on the ecosystem nutritional energy flow principles. Subsequently, diagnostic pigments were employed to represent phytoplankton community biomass, which indicated competition between two groups of phytoplankton in the biochemistry process. Considering empirical relationships between diagnostic pigments and critical environmental factors, biomass responses to river discharge were established by simulating distributions of critical environmental factors under action of river discharges and tide currents. Consequently, environmental flows were recommended for different fish biomass requirements. We used the Yellow River estuary as a case study; and May and June were identified as critical months for maintaining environmental flow. Temporal variation in natural river flow dynamics, which was used as a proxy for environmental flow, should be carefully examined in artificial hydrological regulation strategies, particularly during high-amplitude flood pulses, which might result in negative effects on phytoplankton groups, and subsequently higher aquatic species biomass.