Asparaginase is a therapeutic enzyme used as an anticancer agent and is typically produced through microbial fermentation using organisms such as Escherichia coli and Erwinia chrysanthemi. However, this method faces challenges, including potential enzyme contamination during production, allergic reactions to the enzyme, and stability issues requiring stringent control measures. An innovative solution is the application of plant molecular farming, utilizing Rhizobium root symbiosis for asparaginase production. The objective is to optimize nodule development for asparaginase yield, characterize the enzyme's properties, and evaluate its anticancer efficacy against microbial enzyme. In our study, we established soybean root cultures and inoculated them with Bradyrhizobium japonicum to form root nodules. We evaluated eukaryotic asparaginase production at different incubation times. We purified asparaginase from the root nodule cultures and compared its physicochemical properties and anticancer activity with microbial asparaginase. Results showed that asparaginase reached maximum activity in root nodule cultures 10 days after rhizobium inoculation in the culture media. The root nodule asparaginase exhibited a high content of alpha helices and beta sheets and a low random coil. It demonstrated higher stability and activity across different pH levels and temperatures than Escherichia coli asparaginase. Additionally, root nodule asparaginase displayed better catalytic parameters and stability over time than E. coli asparaginase. Thus, root nodule asparaginase is superior to E. coli asparaginase as an anticancer agent. This ensures the root nodule asparaginase can effectively target cancer cells, enhancing the overall therapeutic outcome. This provides a renewable, cost-effective, and environmentally friendly alternative to traditional enzyme production methods.