Background: Human adipose tissue-derived stem cells (ASCs) are widely used in regenerative medicine and tissue engineering. Magnetic-activated cell sorting (MACS) with monoclonal antibodies (mAbs) that bind surface markers of stem cells such as CD105, CD73, and CD90 is currently applied for the enrichment and isolation of ASCs. Alternatives to mAbs are the variable domains of heavy chain antibodies (VHHs), which are naturally produced in camelids. We report the application of an anti-CD105 VHH conjugated to magnetic beads in the isolation of ASCs in vitro. Results: Two identical anti-CD105 VHHs (EngVHH17) were screened by phage display from a VHH cDNA library constructed from the PBMCs of an alpaca immunized with a lysate of human bladder cancer cell line (T24 cells). EngVHH17 was cloned in pET22b(+), expressed in Escherichia coli BL21 and purified in Ni-NTA chromatography resulting in a ∼15 kDa VHH antibody. EngVHH17 binds CD105 with high affinity (Kd = 3.9 × 10−10 M). The specific binding of EngVHH17 to CD105 was visualized by fluorescence inmunolabeling of phorbol-12-myristate-13-acetate (PMA)-differentiated THP1 cells using FITC labeled anti-6 × His Tag IgG1 mouse mAb. EngVHH17-magnetic beads selectively sorted human ASCs from osteoblasts in a mixed culture in vitro. The selective recovery of ASC cells using different ASC/Osteoblast ratios was higher than 85%. Conclusions: EngVHH17 coupled to magnetic beads binds CD105 expressed on the cell surface of ASCs and isolates them from osteoblasts in mixed cultures in vitro by application of an external magnetic field. EngVHH17 can be further evaluated for the isolation of MSCs by MACS.