There is an ever-growing interest in plant molecular farming as a system for producing valuable recombinant pharmaceutical molecules, such as single-chain variable fragments, on an industrial/agricultural scale and it appears that it is going to become a reality. Human epidermal growth factor receptor-2 (HER2) is an oncogene involved in abnormal cell growth in breast cancer and is considered for the development of new cancer therapies. We describe here the cloning and expression of a scFv-alpha HER2 that has been produced in Escherichia coli and in plants using both stable and transient systems in tobacco and Nicotiana benthamiana. Single-chain antibodies (ScFvs) extracted and purified from E. coli and plant tissues were tested for functionality and specificity by flow cytometry analysis on several cell lines and showed positive results when used on breast cancer slides coming from human frozen tissues. As a result, scFv-alpha HER2 represents a good opportunity for application and use in diagnosis and therapy.