The invention discloses a recombinant large-tooth flounder interleukin-6 protein and a preparation method thereof. The preparation method comprises the following steps: according to a DNA sequence of the large-tooth flounder interleukin-6 protein of paralichthys olivaceus, designing a primer and extracting the total RNA of the kidney fresh by a TRIzol Reagent method, carrying out RT-PCR augmentation, connecting a PCR product and a Pmd19T vector to form a recombinant plasmid, connecting with an expression vector Pet-32a(+) by double digestion, obtaining the recombinant plasmid and transforming the recombinant plasmid into escherichia coli BL21 (DE3); and realizing the soluble expression of the recombinant protein. As the expression process does not generate an inclusion body, the fusion protein for expressing the plasmid has histidine tags, thus being convenient to identification and purification of samples, avoiding the denaturation and renaturation of the samples, having simple operation, reducing the manufacturing cost, and more importantly, having no damage to a protein structure; and the invention has good purity of recombinant protein, high yield and difficult degradation, is applicable to industrial production and lays the foundation of studying the large-tooth flounder interleukin-6 protein of the paralichthys olivaceus.