Assessing the biomass of zooplankton compensates for the difference between number of individuals and the accumulated body weight of the community, which helps assess aquatic ecosystem food web functions. Daphnia are crustaceans that play an intermediate role in biological interactions within food webs. The morphology and body specification of Daphnia differ during growth; hence, it is essential to apply species-specific equations to estimate biomass. We evaluated the length–weight regression equations used previously to estimate Daphnia magna biomass and conducted regression analyses using various body specifications and biomass measurements taken directly using devices such as a microbalance and microscopic camera. Biomass estimated using an equation from the Environmental Protection Agency was significantly different from the direct measurement: average biomass was lower, indicating that the equation possibly underestimated actual biomass. The biomass of D. magna had a higher multiple R2 value when length was compared with width and area, and a linear regression equation was the most suitable equation for biomass estimation. Because body specifications and biomass are affected by various environmental factors, the development of accurate species-specific biomass estimation equations will contribute to obtaining fundamental data with which the biological responses of zooplankton to aquatic ecosystem changes can be assessed.