The accurate magnetoresistance measurement of individual nanostructures is essential and significant for not only enriching the fundamental knowledge of magneto-transport mechanism but also facilitating the designs of desired magnetic nanostructures for diverse technological applications. Herein, we report a deep investigation on the magneto-transport mechanism of single CoCu/Cu multilayered nanowire via direct magnetoresistance measurement by using our invented magnetotransport instrument in-situ scanning electron microscopy (SEM). Off-axis electron holography experiments combined with micromagnetic simulation prove that the CoCu layers in CoCu/Cu multilayered nanowires are formed a multi-domain structure, in which the alignment of magnetic moments is mainly determined by shape anisotropy. The MR of the single CoCu/Cu multilayered nanowire is measured to be only 1.14% when the varied external field is applied along nanowire length axis, which matches with the theoretical prediction of Granular Films model. Density functional theory (DFT) calculations further disclose that spin-dependent scattering at the interface between magnetic and nonmagnetic layers is responsible for the intrinsic magneto-transport mechanism.