The use of long-lived positron emitters 64Cu or 61Cu for labelling of Affibody molecules may improve breast cancer patients' stratification for HER-targeted therapy. Previous animal studies have shown that the use of triaza chelators for 64Cu labelling of synthetic Affibody molecules is suboptimal. In this study, we tested a hypothesis that the use of cross-bridged chelator, CB-TE2A, in combination with Gly-Glu-Glu-Glu spacer for labelling of Affibody molecules with radiocopper would improve imaging contrast. CB-TE2A was coupled to the N-terminus of synthetic Affibody molecules extended either with a glycine (designation CB-TE2A-G-ZHER2:342) or Gly-Glu-Glu-Glu spacer (CB-TE2A-GEEE-ZHER2:342). Biodistribution and targeting properties of 64Cu-CB-TE2A-G-ZHER2:342 and 64Cu-CB-TE2A-GEEE-ZHER2:342 were compared in tumor-bearing mice with the properties of 64Cu-NODAGA-ZHER2:S1, which had the best targeting properties in the previous study. 64Cu-CB-TE2A-GEEE-ZHER2:342 provided appreciably lower uptake in normal tissues and higher tumor-to-organ ratios than 64Cu-CB-TE2A-G-ZHER2:342 and 64Cu-NODAGA-ZHER2:S1. The most pronounced was a several-fold difference in the hepatic uptake. At the optimal time point, 6 h after injection, the tumor uptake of 64Cu-CB-TE2A-GEEE-ZHER2:342 was 16 ± 6%ID/g and tumor-to-blood ratio was 181 ± 52. In conclusion, a combination of the cross-bridged CB-TE2A chelator and Gly-Glu-Glu-Glu spacer is preferable for radiocopper labelling of Affibody molecules and, possibly, other scaffold proteins having high renal re-absorption.