The continuous cultures of the diatom Nitzschia laeviswere performed at different dilution rates ( D) and feed glucose concentrations ( S0) to investigate cellular physiological responses and its production potential of eicosapentaenoic acid (EPA). Steady‐state cell dry weight, residual glucose concentration, cell growth yield, specific glucose consumption rate, and fatty acid profiles were investigated within the range of Dfrom 0.1 to 1.0 day−1( S0fixed at 20 g/L) and the range of S0from 5 to 35 g/L ( Dfixed at 0.3 day−1), respectively. The highest EPA productivity of 73 mg L‐1day‐1was obtained at D= 0.5 day−1and S0= 20 g/L. However, when the continuous culture achieved high productivities of EPA at certain dilution rates and feed glucose concentrations, glucose in the feed could not be consumed completely. Accordingly, the continuous culture was evaluated in terms of both EPA productivity ( P) and glucose assimilation efficiency ( E). The parameter η, defined as the product of Pand E, was used as an overall performance index. Since η is a function of the two independent variables Dand S0, we employed a central composite design to optimize Dand S0for the highest η value. Based on the experimental results of the design, a second‐order polynomial equation was established to represent the relationship between η and Dand S0. The optimal values of Dand S0were subsequently determined as 0.481 day−1and 15.56 g/L, respectively by the empirical model. The verification experiment confirmed the validity of the model. Under the optimal conditions, η value reached 46.5 mg L‐1day‐1, suggesting a considerably high efficiency of the continuous culture of N. laevisin terms of EPA production and glucose utilization.