The continuous cultures of the diatom Nitzschia laevis were performed at different dilution rates (D) and feed glucose concentrations (S-0) 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 D from 0.1 to 1.0 day(-1) (So fixed at 20 g/L) and the range of S-0 from 5 to 35 g/L (D fixed at 0.3 day(-1)), respectively. The highest EPA productivity of 73 mg L-1 day(-1) was obtained at D = 0.5 day(-1) and S-0 = 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 eta, defined as the product of P and E, was used as an overall performance index. Since eta is a function of the two independent variables D and S-0, we employed a central composite design to optimize D and S-0 for the highest eta value. Based on the experimental results of the design, a second-order polynomial equation was established to represent the relationship between eta and D and S-0. The optimal values of D and S-0 were subsequently determined as 0.481 day(-1) and 15.56 g/L, respectively by the empirical model. The verification experiment confirmed the validity of the model. Under the optimal conditions, eta value reached 46.5 mg L-1 day(-1), suggesting a considerably high efficiency of the continuous culture of N. laevis in terms of EPA production and glucose utilization.