Objective To establish and validate a simple and economical acetonitrile precipitation method to deplete albumin from serum and investigate its effectiveness. Methods The albumin was precipitated by mixing delipidated serum, water, acetonitrile at ratio of 1∶2∶4.5 (v/v/v). The removal effects and repeatability were analyzed by onedimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The gel images of serum samples from 4 healthy controls and 4 patients with nonsmallcell lung carcinoma were analyzed before and after treatment. Results The results showed that the average concentration of albumin in the sera was decreased from (50.65 ± 2.82) g/L to (2.50 ± 0.71) g/L. The percentage of albumin in the total protein of serum was decreased from 65.1% to 33.3%, and significantly statistical differences were found (P<0.01). Compared with the gel images of the original serum with same volume, the band of protein with molecular weight between 50-80 kDa was removed. The band narrowed significantly and low abundance proteins could be seen. Conclusions The acetonitriledepletion strategy may offer an economical, simple and reproducible method to remove albumin from human serum, which provided a technical support for further study of serum proteomics.