Pseudomonas aeruginosa isolate JY21 (GenBank accession number, KF922508) which produces pyocyanin was tested as biocontrol agent for its antagonistic effect on the in vitro growth of plant pathogens namely, Alternaria sp, Pythium sp, Phytophthora infestans, Rhizoctonia solani and Sclerotium sp. Sequential optimization strategy, based on statistical experimental designs, was implemented for enhancing production of pyocyanin as biocontrol agent. For screening of various fermentation factors significantly influencing pyocyanin production, Plackett–Burman design was used. Fourteen different factors (variables) with two-level including fermentation conditions and medium constitution were chosen to perform this optimization process. pH, KCl and MgCl2.6H2O were found to be the most significant variables affecting pyocyanin concentration. A near optimum medium formulation was obtained using this method, pyocyanin concentration achieved on this medium was 217.5 µg/ml. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, Box–Behnken design with three-level was employed. The optimal levels of the three variables as obtained from the maximum point of the polynomial model were estimated using the Solver function of Microsoft Excel tools and JMP program, and found to be: pH 8.2, KCl, 1.25 g/l and MgCl2.6H2O, 4.2 g/l with a predicted pyocyanin concentration of 245.39 µg/ml.