OBJECTIVE: To compare the efficacy and safety of different asparaginase formulations in the treatment of acute lymphoblastic leukemia (ALL) based on nano-magnetic bead immunoassay. METHODS: Retrospective analysis of adult ALL patients' clinical data who admitted to The Affiliated Hospital of Changsha Health Vocational College from August 2020 to August 2023. Finally, 65 adult ALL patients were included in this study, including the polyethylene glycol conjugated asparaginase (PEG-ASNase) group (n = 32) and the L-asparaginase (L-ASNase) group (n = 33). Enzyme-linked immunosorbent assay (ELISA) based on magnetic nanoparticles was used to determine the activity of ASNase in both groups. The levels of asparagine or glutamine in two groups were detected by automatic biochemical analyzer during induction therapy, and the adverse events of the two groups were observed during the treatment. RESULTS: PEG-ASNase demonstrated a slower decrease in enzyme activity, longer action duration, and higher safety profile compared to L-ASNase. PEG-ASNase group and L-ASNase group demonstrated a similar complete remission rate (71.88% vs. 60.61%). Event-free survival was higher in patients receiving PEG-ASNase than those receiving L-ASNase (42.4% and 18.7%). The observed adverse reactions included allergic reactions, pancreatic lesions, gastrointestinal reactions and liver function damage. The incidence of gastrointestinal reactions and liver function damage was higher in the L-ASNase group than that in PEG-ASNase group (45.45% and 33.33%). CONCLUSION: This study provides valuable insights into the asparaginase treatments in clinical, highlighting the importance of PEG-ASNase for improving treatment protocols in adult ALL patients.