Hemoglobin, which widely exists in all vertebrates and in some invertebrates, is possibly a precursor of antimicrobial peptides (AMPs). However, AMPs in the hemoglobin of invertebrates have been rarely investigated. This study is the first to report the full-length cDNA, prokaryotic expression, and antimicrobial activity of UuHb-F-I from Urechis unicinctus. The full-length cDNA sequence of UuHb-F-I was 780 bp with an open-reading frame of 429 bp encoding 142 amino acids. MALDI-TOF-MS suggested that the recombinant protein of UuHb-F-I (rUuHb-F-I) yielded a molecular weight of 15,168.01 Da, and its N-terminal amino acid sequence was MGLTGAQIDAIK. rUuHb-F-I exhibited different antimicrobial activities against microorganisms. The lowest minimum inhibitory concentration against Micrococcus luteus was 2.78–4.63 μM. Our results may help elucidate the immune defense mechanism of U. unicinctus and may provide insights into new AMPs in drug discovery.