<p>This study focused on phytase overproduction in recombinant <i>E. coli</i> BL21(DE3)/pET-<i>appA</i> expressing <i>E. coli</i> phytase(APPA). A modified mineral salt medium was investigated for the strain growth and phytase production in shake-flask, which included 30&#xa0;mM NH₄OH and 17&#xa0;g L⁻¹ KH₂PO₄ as sole nitrogen(N) and phosphorous(P) sources, respectively. After that, fed-batch cultivation process, especially induction strategy feeding lactose intermittently was optimized in 30L bioreactor by Taguchi orthogonal experiments (L₁₈(2¹ × 3⁶)) at an induction cell density of OD₆₀₀ 25, followed by research on effect of induction cell densities. The optimized fed-batch cultivation process parameters included glucose starvation time of 15&#xa0;min just before induction, induction cell density of OD₆₀₀ 45, induction temperature of 25&#xa0;°C, predetermined specific growth rate of 0.04&#xa0;h⁻¹, DO level of 15%, lactose concentration of 60&#xa0;mM, lactose feed times of 6 times, and pH of 7.2–7.6. In the optimized condition, the highest soluble phytase activity of 2 152U&#xa0;mL⁻¹ ever reported was obtained at 33.0&#xa0;h, indicating the enhancements of 31.6-fold and 3.7-fold compared to batch cultivation process in shake flask and pre-optimized fed-batch cultivation process in the bioreactor, respectively. We propose that the defined medium, the induction strategy and fed-batch process parameters could be applied to overproduce soluble proteins in <i>E. coli.</i></p>