<p>The current study aims to gain a deeper understanding of the design and optimization of streptokinase-loaded nanoparticle systems using the Box-Behnken Design (BBD). The outcomes can further be applied to improve the oral bioavailability of streptokinase.&#xa0;Thrombolytic therapy plays a vital role in the treatment of thrombotic diseases. Streptokinase, a plasminogen activator, has been widely used for its low cost and efficient thrombolysis. As it is a protein, it is mostly administered parenterally, and the current study focuses primarily on the oral bioavailability of Streptokinase.&#xa0;The nanoparticulate system is prepared using a coacervation process using the polymers chitosan and alginate loaded with streptokinase. The nanoparticles prepared were characterised by using Zetasizer, FESEM, UV Spectrophotometer, and FTIR. The nanoparticulate system was further optimised by Box-Behnken Design (BBD) for different factors like Streptokinase concentration, polymer concentrations, and stirring speed on parameters such as particle size and drug loading. The in vitro release profile of Streptokinase from nanoparticles was examined to estimate its potential for increased bioavailability.&#xa0;The optimized formula projected by the software Design-Expert® is 1.165 mg/mL SK, 0.500 mg/mL sodium alginate, and a stirring speed of 875.42 rpm. The particle size and loading were 282.60 nm and 26.01%, respectively, indicating excellent model desirability. Response surface research indicated that SK and alginate have strong interaction effects on particle size and loading efficiency. The improved formulation shows potential for excellent delivery with controlled particle properties. The maximal in vitro release from optimised streptokinase-loaded nanoparticles was 58.04% in PBS.&#xa0;The optimised streptokinase-loaded nanoparticulate system prepared using chitosan and sodium alginate can be a novel delivery method for streptokinase and similar biological products.</p> Graphical Abstract <p></p>