Positively charged mitoxantrone (MTO) was absorbed by negatively charged blank bovine serum albumin (BSA) and chitosan (CS) nanospheres to form MTO-BSA-NS and MTO-CS-NS, respectively. In addition to other conditions, values of pH of every step were optimized. On optimized conditions, MTO-BSA-NS of a mean size of 77 nm with an encapsulation yield (EY) of (98.86+/-1.43)% [drug loading rate (DL) (19.82+/-0.29)%] and MTO-CS-NS of a mean size of 75 nm with an EY of (97.57+/-1.00)% [DL (9.78+/-0.10)%] were obtained. After lyophilization and sterilization by (60)Co, the mean size increased about 10% but no significant change was observed in EY and DL. Tests for in vitro release in physiological saline or physiological saline containing 0.5% (w/v) ascorbic acid by a dialysis bag showed sustained release and little burst effect.