Quantum dots (QDs) have been widely used for bioimaging in vivo because of their excellent optical properties. As part of the preparation process of QD-based nanohybrids, purification is an important step for minimizing contaminants and improving the quality of the product. In this work, we describe high-performance size exclusion chromatography (HPSEC) used to purify nanohybrids of CdSe/ZnS QDs and anti-human epidermal growth factor receptor 2 antibodies (QD-HER2-Ab). The unbound antibody and suspended agglomerates were removed from freshly prepared QD-HER2-Ab via HPSEC. Pure and homogeneous QD-HER2-Ab were then used as immunofluorescence target imaging bioprobes in vivo. The QD-HER2-Ab did not cause any obvious acute toxicity in mice one week after a single intravenous injection of 15 nmol/kg. The purified QD-HER2-Ab bioprobes showed high tumor targeting ability in a human breast tumor xenograft nude mouse model (24 h after injected) with the possibility of in vivo immunofluorescence tumor imaging. The immunofluorescence imaging background signal and acute toxicity in vivo were minimized because of the reduction of residual QDs. HPSEC-purified QD-HER2-Ab is an accurate and convenient tool for in vivo tumor target imaging and HER2 detection, thus providing a basis for the purification of other QD-based bioprobes.