In order to study the expression of PKCθ and Spartin in Nile tilapia (Oreochromis niloticus), these two proteins were expressed in Escherichia coli, purified, and then used to immunize Japanese big-ear white rabbits (Oryctolagus cuniculus) according to the conventional method to prepare rabbit anti-PKCθ and rabbit anti-Spartin polyclonal antibody. The titers of rabbit anti-PKCθ and rabbit anti-Spartin antiserum were evaluated by indirect ELISA. The expressions of these two proteins in different issues (liver, spleen, intestine and muscle) of Nile tilapia were detected by Western Blotting. The results showed that the prokaryotic expression vectors of pET-B2m-PKCθ and pET-B2m-Spartin were successfully constructed, the recombinant PKCθ and Spartin proteins were expressed in inclusion body with molecular weight of 56 ku and 30 ku, respectively. The indirect ELISA assay showed that the rabbit anti-PKCθ and rabbit anti-Spartin antisera had a good sensitivity with the titer of 1︰512 000, and Western blotting showed that the polyclonal antibody had good specificity. PKCθ protein was expressed in the liver, spleen, intestine and muscle. Spartin protein was only expressed in the muscle. In this study, the PKCθ and Spartin recombinant proteins of Nile tilapia were expressed and purified successfully, the polyclonal antibodies against PKCθ and Spartin proteins with a high titer were obtained, and the expressions of PKCθ and Spartin in different issues of Nile Tilapia were determined. The current study will shed new light on the functional and mechanism study on PKCθ and Spartin in Nile tilapia.