As an excellent reporter molecule, enhanced green fluorescent protein (eGFP) was widely used for gene expression and regulation and was generally expressed in Escherichia coli strain. A rapid procedure consisting of ammonium sulfate precipitation, size exclusion chromatography, and anion exchange chromatography was developed for the purification of eGFP. Based on the proposed procedure, recombinant eGFP with an electrophoretic purity was achieved in combination with an overall yield of 66% and a purification factor of 17.9. The fluorescent spectrometry of purified eGFP and lysate from E. coli strain expressing eGFP exhibited the same wavelength of excitation and emission maxima, indicating that the purification procedure did not influence the construct and fluorescent characteristics of desired protein. The procedure mentioned was easy to scale up for the purification of large quantities of eGFP.