Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6) cfu/3 µg and 2×10(9) cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.