The purpose of this study was to establish a novel nontoxic disulfide bond reducing method for lipophilic drug-loaded albumin nanoparticle preparation and make a systematic investigation on this method. Cysteine (Cys) was used to break the disulfide bond of albumin and introduce the self-assembly of drug and albumin. Paclitaxel (PTX) and bovine serum albumin (BSA) were selected to be the model lipophilic drug and albumin. The particle formation dynamics, influencing factors and formation mechanisms were investigated by determining the characteristics of particles including the particle size and yield. Nanoparticles with diameter of 50–400nm and drug loading efficiency up to 18.3% were prepared successfully. pH 7–8 was suitable for nanoparticle preparation. Temperature, BSA concentration and Cys concentration had positive effects on the particle size and yield. When PTX added was less than the maximal amount of PTX that could bind to BSA, particles with a spherical structure could be formed; otherwise nanoparticles with a core–shell structure could be formed. This novel nontoxic disulfide bond reducing method provides a common and safe method for preparing various kinds of albumin-based nanocarriers for a wide range of applications, from drug (especially the lipophilic drug) delivery to diagnosis of disease.