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 Cu2+. We synthesized autofluorescent microspheres crosslinked with glutaraldehyde by controlling volume ratio between oil phase and water phase. A novel fluorescent platform for sensing Cu2+ based on the autofluorescence quenching of PEI microspheres by Cu2+ with a detection limit as low as 2 nM. In addition, the absorbance of PEI microspheres at 205 nm, 325 nm, 590 nm and 800 nm were sensitive to the presence of Cu2+. The strong chelation interaction between Cu2+ 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 Cu2+ would provide autofluorcent microspheres a future application in quantitative analysis.