In this paper, an original strategy is employed to biosynthesize the isoprene by heterologously co-expressing the Saccharomyces cerevisiae MVA pathway and isoprene synthase (IspS) from Populus alba in the Escherichia coli BL21 (DE3) strain, which was screened from three different IspS enzymes. The finally genetic strain YJM13 harboring the MVA pathway and ispSPa gene could accumulate isoprene up to 2.48 mg/l and 532 mg/l under the flask and fed-batch fermentation conditions, respectively, which is about three times and five times to the control strain. The result proves to be higher than that in the report documents. In this way, a potential production system for isoprene from renewable sources via the MVA pathway in E. coli has been provided.