Hemoglobin, composed of α- and β-chains, is essential for oxygen transport and is key in diagnosing and treating gastrointestinal and blood disorders. It also aids in detecting blood contamination and estimating transfusion volumes. Immunological methods, based on antigen-antibody interactions, are distinguished by their high sensitivity and accuracy. Consequently, it is necessary to develop hemoglobin-specific antibodies characterized by high specificity and affinity to enhance detection accuracy. The variable domain of the new antigen receptor (VNAR) from sharks, the smallest antigen-binding unit, is ideal for disease diagnosis and treatment due to its small size, stability, and high affinity. In this study, Chiloscyllium plagiosum was immunized with human hemoglobin protein. Nine VNAR immune libraries with sizes ranging from 1 × 10