The extensive applications of engineered nanomaterials (ENMs) can result in their release into waters. Membrane processes have great potential in reducing ENMs release. In that case, the issues of particular concern are membrane fouling caused by ENMs during separation processes. The objective of the present study is to improve the understanding of how ENMs are retained by membranes. An innovative methodology using confocal laser scanning microscopy (CSLM) is developed to locate fluorescent CdTe quantum dots (QDs) in different depths of fouled ultrafiltration membranes. With the help of image analysis software, both qualitative and quantitative information about the distribution of QDs in membranes are obtained. For low molecular weight cut off (MWCO) membranes (1, 5 and 10 kDa), QDs (sizes from 1 to 5 nm) distributed mainly around surfaces or on top of membranes, accompanied with near 100% retention regardless of transmembrane pressure. As membrane MWCO increased (30 and 100 kDa), more QDs could pass through membranes accompanied with decreasing retention efficiencies and the occurrence of QDs was usually in deeper positions of membranes. Distribution results were in agreement with fouling analysis which demonstrated that standard blocking (internal fouling) and/or cake models (external fouling) frequently occurred during filtrations of QDs.