Abstract: Objective To determine the pharmacokinetics and bioequivalence of self-assembled hyaluronic acid-graft-poly (ethylene glycol)/hydroxypropyl-beta-cyclodextrin hollow nanospheres loaded with asparaginase (AHHPs) in SD rats. Methods AHHPs were prepared and observed under transmission electron microscopy. Its size, Zeta potential and entrapment efficiency were detected. Asparaginase (AAS) activities were measured after intravenous injection of AHHPs or free AAS in rats. The pharmacokinetic parameters were calculated using software DAS 2.1.1 for bioequivalence assessment. Results The self-prepared AHHPs had an average particle size of (367.43±2.72) nm, Zeta potential of (-15.70±1.25) mV, and average entrapment efficiency of (66.03 ± 3.81)%. The intravenous injection of AHHPs and free AAS produced an area under concentration-time Curve (AUC)(0-48 h) of (162.06±4.01) U/mL·h and (46.38±1.98) U/mL·h, AUC(0-∞) of (203.74±12.91) U/mL·h and (51.44±3.01) U/mL·h, mean residence time (MRT)(0-72 h)of (4.35±0.06) h and (1.76±0.06) h, MRT(0-∞) of (7.53±1.05) h and (2.44±0.29) h, peak concentration (Cmax) of (30.37±0.43) U/mL and (26.06±0.88) U/mL, and time to peak concentration (Tmax) of (0.75±0.00) h and (0.08±0.00) h, respectively. Compared with free AAS, the AUC(0-48 h), AUC(0-∞), MRT(0-72 h), MRT(0-∞), Cmax and Tmax of AHHPs increased by 3.5, 4.0, 2.5, 3.1, 1.2 and 9.4 times, respectively. The 90% confidential intervals of AUC(0-48 h), AUC(0-∞) and Cmax of the tested formulation were 72.6%-74.0%, 72.3%-73.7%, 94.7%-96.3%, respectively. Conclusion AHHPs can improve the bioavailability of AAS, extending its biological half-life in rats. AHHPs and free AAS are not bioequivalent.