Interleukin 24 (IL-24) has a broad spectrum of specific antitumor activities without affecting normal cells. The recombinant human IL-24 (rhIL-24) expressed in E. coli has low biological activity due to lack of necessary glycosylation modification. In this study, based on the modification of the non-glycosylated IL-24 with polyethylene glycol (PEG), we aimed to improve the stability and prolong its half-life in vivo. Firstly, the recombinant plasmid containing the hIL-24 cDNA was prepared by the prokaryotic-expression plasmid pET-28a and transformed into E. coli BL21. After induced by isopropyl beta-D-thiogalactoside (IPTG), the target protein rhIL-24 was expressed as insoluble inclusion body, which was solubilized and denatured by 6 M guanidine hydrochloride. The denatured rhIL-24 was diluted to refold in the optimized buffer overnight at the protein concentration of 0.1 mg/mL. The refolded rhIL-24 was mainly in the form of soluble aggregate, but high-purity monomer rhIL-24 was obtained through size exchange chromatography with the addition of SDS in elution buffer. The tertiary structure of rhIL-24 was confirmed by fluorescence spectroscopy. Western blot analysis showed that rhIL-24 could be site-specifically modified by mPEG5000-ALD. Methyl thiazolyl tetrazolium (MTT) assay showed no significant difference between mPEG5000-ALD-rhIL-24 and rhIL-24 in inhibiting the growth of melanoma cell line A375 in vitro. Pharmacokinetic studies showed that PEG modification could significantly improve the stability and prolong the half-life of rhIL-24 from 8.41 to 13.2 h. The data strongly suggested that mPEG-ALD 5000 could site-specifically modify rhIL-24 expressed in E. coli. The PEG modification significantly prolonged the half-life of rhIL-24 without reducing its antitumor activity in vitro.