Antibody-based therapies offer significant promise for the treatment of various diseases, including cancer and infectious agents. In cancer immunotherapy, these therapies aim to activate the immune system against tumor cells or inhibit their growth. However, the complex nature of cancer necessitates the exploration of multi-target therapeutic approaches, such as bispecific antibodies. These antibodies offer the potential to target multiple cancer types and variants, potentially overcoming resistance mechanisms. Single-domain antibodies (sdAbs) possess unique characteristics, including a compact size, high stability, and specific antigen recognition, making them ideal building blocks for bispecific antibody development. In this study, we describe the development of a readily manageable and cost-effective sdAb discovery platform utilizing rabbit immunization. Briefly, rabbits were immunized with a recombinant CD3 epsilon domain to elicit an immune response. Subsequently, antigen-specific sdAbs were isolated from the immunoglobulin heavy chain variable region. This accessible sdAb discovery platform has the potential to be applied in a variety of fields, including biological research and therapeutic development.