<p>Background: Expression of heterologous proteins at large scale is often a challenging job due to plasmid instability, accumulation of acetate and oxidative damage in bioreactors. Therefore, it is necessary to optimize parameters influencing cell growth and expression of recombinant protein.&nbsp;&nbsp;<br /> Methods: In the present study, the optimal culture conditions for expression of reteplase by <em>Escherichia coli (E. coli)</em> BL21 (DE3) in a bench-top bioreactor was determined. Response Surface Methodology (RSM) based on Box-Behnken design was used to evaluate the effect of three variables (<em>i.e</em>., temperature, shaking speed and pH) and their interactions with cellular growth and protein production. The obtained data were analyzed by Design Expert software.<br /> Results: Based on results of 15 experiments, a response surface quadratic model was developed which was used to explain the relation between production of reteplase and three investigated variables. The high value of &quot;R-Squared&quot; (0.9894) and F-value of 51.99 confirmed the accuracy of this model. According to the developed model, the optimum fermentation conditions for reteplase expression were temperature of 32&deg;<em>C</em>, shaking speed of 210 <em>rpm</em>, and pH of 8.4. This predicted condition was applied for the production of reteplase in the bioreactor leading to a protein yield of 188 <em>mg/l</em>.<br /> Conclusion: Our results indicate the significant role of culture conditions (<em>e.g</em>., pH, temperature and oxygen supply) in protein expression at large scale and confirm the need for optimization. The proposed strategy here can also be applied to experimental set-up of optimization for fermentation of other proteins.</p>