Apolipoprotein E (ApoE)’s ϵ4 alle is the most important genetic risk factor for late onset Alzheimer’s Disease (AD). Cell-surface heparan sulfate (HS) is a cofactor for ApoE/LRP1 interaction and the prion-like spread of tau pathology between cells. 3-O-sulfo (3-O-S) modification of HS has been linked to AD through its interaction with tau, and enhanced levels of 3-O-sulfated HS and 3-O-sulfotransferases in the AD brain. In this study, we characterized ApoE/HS interactions in wildtype ApoE3, AD-linked ApoE4, and AD-protective ApoE2 and ApoE3-Christchurch. Glycan microarray and SPR assays revealed that all ApoE isoforms recognized 3-O-S. NMR titration localized ApoE/3-O-S binding to the vicinity of the canonical HS binding motif. In cells, the knockout of HS3ST1-a major 3-O sulfotransferase-reduced cell surface binding and uptake of ApoE. 3-O-S is thus recognized by both tau and ApoE, suggesting that the interplay between 3-O-sulfated HS, tau and ApoE isoforms may modulate AD risk.