Hepcidin is a kind of cysteine-rich antimicrobial peptide that plays a vital role in host innate immune activity and iron regulation. Here, we report the molecular characterization and functional analysis of a novel hamp1 hepcidin isoforms Tf-Hep from roughskin sculpin, Trachidermus fasciatus. A cDNA fragment of 988 bp with an ORF of 273 bp was obtained. The coding sequence encodes for a signal peptide of 24 amino acids coupled with a prodomain of 40 amino acids and a mature peptide of 26 amino acids. Tissue distribution analysis indicated that Tf-Hep was most abundant in the liver. It could be significantly induced post lipopolysaccharide (LPS) challenge and heavy metal exposure. The mature peptide was expressed as a 6.061 kDa fusion protein in Pichia pastoris GS115. The active purified recombinant protein (rTf-Hep) exhibited a wide spectrum of potent antimicrobial activity in vitro against 4 Gram-negative bacteria Escherichia coli, Vibrio Anguillarum, Klebsiella pneumoniae, and Pseudomonas aeruginosa and 4 Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus megaterium with minimum inhibitory concentrations (MICs) of 5–80 μg/ml (0.825–13.2 μM). It also displayed high affinity to polysaccharides on bacteria surface including LPS, lipoteichoic acid (LTA) and peptidoglycan (PGN). We further revealed that rTf-hep was capable of agglutinating 6 of the 8 bacteria. All these results suggest that rTf-hep may be both an antibacterial effector and a pattern recognition molecule in fish immune defense. The in vivo bacterial treatment results demonstrated that rTf-Hep could significantly improve the survival rate of fish infected with V. anguillarum. Taken together, these data indicate an important role for Tf-hep in the innate immunity of Trachidermus fasciatus and suggest its potential application in aquaculture for increasing fish resistance to disease.