The considerable effect of enzymes on human health draws great attention to enzyme-based drugs (therapeutic enzymes), in recent times. L-asparaginase (ASNase) is a well-known therapeutic enzyme. It has varied applications and is a single molecule for the treatment of multiple diseases. This study tries to extract asparaginase from soybean debris (agricultural wastes) as a cheap plant source and compare this with microbial asparaginase as an agent in cancer chemotherapy. The asparaginase was extracted and purified from soybean debris (plant asparaginase) and Pseudomonas aeruginosa (microbial asparaginase), then the physiochemical characters were determined for the two enzymes, and the anticancer activity of plant and microbial asparaginase was determined against gastric cancer (CLS-145), pancreatic cancer (AsPC-1), colon cancer (HCT116), esophagus cancer (KYSE-410), liver cancer (HepG2), breast cancer (MCF-7), and cervical cancer (HELLA). The results showed that plant asparaginase was superior to microbial asparaginase in its physiochemical characters. Plant asparaginase showed higher stability and activity under the conditions of changing either the temperature or the pH; also plant asparaginase has a higher affinity to the asparagine than the microbial asparaginase; besides, this plant asparaginase did not show activity with glutamine as a substrate. The plant asparaginase showed higher anticancer activity than that of microbial asparaginase against all studied cancer cell lines. The present study introduces as the first time a comparative study between the plant and microbial asparaginase which proves that soybean debris asparaginase can be more efficient and safe than that of the microbial asparaginase as an anticancer agent.