Using pure human serum albumin (HSA) as the model protein, the effects of protein on the extraction of antipsychotic drugs (APDs: diazepam, chlorpromazine hydrochloride, and perphenazine) in human serum sample were studied. The present paper investigated the interaction between APDs and HSA by fluorescence spectrometry in detail. The influences of different ethanol concentration solution on protein denaturation were studied by Rayleigh scattering. The results showed that APDs strongly bound with HSA. In the φ(ethanol) 80% extracting solution, a slow but full protein denaturation takes place, which causes the unfolding of protein and the dissociation of drugs. Then K2HPO4 was added into the extracting solution to form aqueous two-phase system, and meanwhile the drug residues were extracted into upper phase with high extraction efficiencies. After filtration, the upper phase was ready for analysis of drug residues by HPLC system. The detection limits were in the range of 18.8~38.4 ng·mL-1, and the spiked recovery was 94.2%~98.7% for determination of antipsychotic drugs in human serum. The method is efficient, solvent-saving, environment-friendly, and accurate.