<p>Serratiopeptidase, a fibrinolytic and anti-inflammatory biological macromolecule is in high demand for its therapeutic applications. Thus, exploration of novel enzyme sources with high-yielding serratiopeptidase fermentation capability is necessary. The present study attempts to maximize serratiopeptidase synthesis from <i>Serratia marcescens</i> SP6 by optimizing the fermentation conditions using sequential strategy of experimental designs. Of the 11 fermentation parameters evaluated under one-factor-at-a-time approach and Plackett–Burman design, 3 parameters namely dextrose, casein, and initial pH of medium significantly influenced serratiopeptidase production (<i>p</i> &lt; 0.0001). These were evaluated further by the central composite rotatable design of response surface methodology to determine the effects of their interaction on serratiopeptidase production. Post-optimization, 2489.71 U/mL was predicted as the maximum serratiopeptidase activity at conditions of 10&#xa0;g/L of casein, 18.40&#xa0;g/L of dextrose, and pH 7 of fermentation broth. Upon experimental evaluation of the assay conditions, 2495 U/mL of serratiopeptidase activity was recorded which is in good agreement with the predicted response. An overall increase of 10.16-fold in the enzyme production was thus achieved from the unoptimized condition. The findings from this sequential optimization method of enzyme synthesis demonstrated the applicability of the predicted optimum conditions for sustainable large-scale serratiopeptidase production from <i>Serratia marcescens</i> SP6.</p> Graphical Abstract <p></p>