Bacterial hemoglobin from Vitreoscilla (VHb) is recognized as a good fusion protein for the soluble expression of foreign protein. In this study, we generated a monoclonal antibody (MAb) against VHb for its detection. For the rapid screening of MAb, a protein chip technology based on the Alexa-488 (A488) dye labeling method was introduced. In order to fabricate the chip, the VHb protein was chemically coupled to the chip surface and then the culture supernatants of 84 hybridoma cell lines were spotted onto the VHb chip. The bound MAbs were measured by A488-modified anti-mouse IgG. A single spot (MAb A10) exhibited significantly high signal intensity. The immunoblot analysis evidenced that the MAb A10 can detect VHb-fused proteins with high specificity.