In this paper, an autofluorencent polyethyleneimine (PEI) microspheres probe has been designed as the two channel sensor for both quantitative fluorescence and visual detection of Cu(2+). We synthesized autofluorescent microspheres crosslinked with glutaraldehyde by controlling volume ratio between oil phase and water phase. A novel fluorescent platform for sensing Cu(2+) based on the autofluorescence quenching of PEI microspheres by Cu(2+) with a detection limit as low as 2nM. In addition, the absorbance of PEI microspheres at 205nm, 325nm, 590nm and 800nm were sensitive to the presence of Cu(2+). The strong chelation interaction between Cu(2+) and -NH2 on the surface of microspheres produced the fluorescence quenching and color change of PEI microspheres which facilitated our sensing approach. We regard the developing of autofluorcent PEI microspheres as the dual-channel sensor for Cu(2+) would provide autofluorcent microspheres a future application in quantitative analysis.