Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy-chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy-chain antibody (VHH) or nanobody. The very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent, so the anti-nanobody HRP conjugate is one of the key components in production, characterization, and application of these molecules in detection and therapy. These antigen-specific fragments are well expressed in Escherichia coli. Here we report high expression and purification of some nanobodies against tumor markers. The nanobody genes were subcloned into a pET22b(+) vector to overexpress the protein coupled with fusion tag in E. coli. The expressed nanobodies were purified by immobilized metal affinity chromatography and injected into rabbits as an immunogen. Described here are the preparation, purification, and characterization of anti-nanobody HRP conjugate for use in the various nanobody immunoassay systems. Several biochemical modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product. We concluded that the present reagent can detect nanobodies in various detection procedures with great sensitivity and accuracy.