The study aimed to purify and characterize the enzyme L-asparaginase from Lactobacillus rhamnosus to produce L-asparagine, Lactobacillus rhamnosus was cultured using liquid media, fermentation, and then the Nesslerization method was used. Lactobacillus rhamnosus is a probiotic bacteria strain isolated of the mother's humans breast milk. It has been shown to inhibit the growth of pathogens, This bacteria modulates the immune system and has severe anti-inflammatory effects.The isolates bacteria were recognize build on various tests, including cultural, morphological, chemical, and API 50 to recognize isolates Lactobacillus rhamnosus. Through the apparent results, it was shown that the bacterial isolates produced the L-asparginase enzyme on the modified medium and the color changed from yellow to red as a positive result in the presence of phenol red dye as an indication of the result.However, the isolates bacteria showed the highest of enzyme production with pink inhibition zones of approximately 14.81 mm after two days and 99.137 IU/mg of specific activity.In this study, the pH and temperature were evaluated for the production of L-asparaginase enzyme and the maximum production was at pH 6.5 and at a temperature of 35°C. Investigations have indicated that L-asparaginase in the future may have an important and essential role in uses clinical and its applications in clinical