Abstract. Over the past few decades, there has been considerable interest in developing protein nanoparticles as drug delivery devices. The underlying rationale is their exceptional characteristics, namely biodegradability and nonantigenicity. Herein, phase separation method was used to prepare 5-fluorouracil-loaded bovine serum albumin (BSA) nanoparticles. Drug release was tracked by continuous flow dialysis technique. Effect of process variables on loading efficiency of 5-fluorouracil was investigated and optimized through Taguchi's M16 design with the amount of entrapped drug as response. Optimum condition was found to be 2 mg/mL of 5-fluorouracil, 3.7 mL of added ethanol, 176 µL of glutaraldehyde, drug-protein incubation time of 30 min, and pH of 8.4 for 200 mg of BSA in 2 mL drug solution. pH had the most noticeable effect on the amount of entrapped drug, but glutaraldehyde had the least. Mean diameter and zeta potential of fabricated nanoparticles under these conditions were 210 nm and −31.7 mV, respectively. Drug-loaded BSA nanoparticles suspension maintained constant release of drug for 20 h under experimental conditions, so this colloidal drug carrier is capable of releasing drug in a sustained manner.