In order to find a simple, fast and highly efficient method for the separation and purification of nerve growth factor (NGF) from venom of Chinese Cobra, the combination process of several different kinds of chromatographic media was studied. According to the properties of NGF and the character of different chromatographic media, a novel two-step chromatographic purification method, consisting of chromatography of crude venom on DEAE-Sepharose F. F. anion-exchange medium followed by a size exclusion on a Sephadex G-50 column, is presented. The DEAE-Sepharose F. F. chromatographic column was 20 cm x 3.5 cm i.d. and first eluted with 50 mmol/L Tris-HCl (pH 8.5) for 50 min, and separately followed by an elution with a linear gradient of 0 - 200 mmol/L NaCl containing 50 mmol/L Tris-HCl (pH 8.5) for 190 min and with 200 mmol/L NaCl containing 50 mmol/L Tris-HCl (pH 8.5) for 60 min. The flow rate of mobile phase was 6.1 mL/min. The Sephadex G-50 column was 150 cm x 3.5 cm i.d. and eluted with 50 mmol/L phosphate-buffered saline (PBS) (pH 7.5) for 1 250 min. The flow rate of elution solution was 2.4 mL/min. Through this two-step chromatographic purification process, the obtained NGF was proved to be homogeneous on sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and its relative molecular mass was estimated to be approximately 29 000, which was consistent with that reported in literature. On a Shim-Pack VP-ODS reversed-phase high performance chromatographic column (4.6 mm i.d. x 150 mm), its purity was about 98.3%. The total protein recovery of this purification method was 0.51% and the obtained NGF had the activity of eliciting neurite outgrowth from chick embryonic dorsal root ganglia. This two-step chromatographic process is simple and highly efficient. It can be used to isolate and purify NGF from venom of Chinese Cobra in large-scale.