Objective To prepare hyaluronic acid-graft-poly (ethylene glycol)/α-cyclodextrin hollow nanocapsules loaded with asparaginase (AHAPs) and to investigate their in vitro stability and activity. Methods We prepared the AHAPs by self-assembly method and detected the optimal temperature, optimal pH value, particle size, zeta potential and entrapment efficiency. Then the differences in in vitro stability between AHAPs and free asparaginase (AN) were investigated by measuring thermal stability, acid- and basic-stability, stability to trypsinase, stability to metal ions and organic compounds, plasma stability and storage stability. The interaction between AN and blank hyaluronic acid-graft-poly (ethylene glycol)/α-cyclodextrin hollow nanocapsules was studied through the fluorescence experiment. Results The optimal temperature for AHAPs was 50 ℃, the optimal pH value was 7.0, the mean particle size was (424.53±7.25) nm, and the mean zeta potential was (－48.77±0.99) mV. The entrapment efficiency of AHAPs was (64.40±1.82)%. The results of stability experiment showed that AHAPs had a significantly better in vitro stability than free AN, and some stability experimental results were statistically significant (P<0.05). Fluorescence experiment showed that the improvement of biological activity of AHAPs may be related to the change of protein residues microenvironment and enzyme conformation caused by the interaction between AN and blank hyaluronic acid-graft-poly (ethylene glycol)/α-cyclodextrin hollow nanocapsules. Conclusion It has been found that AHAPs can not only improve the activity but also greatly enhance the stability of AN in vitro.