Gonadotropin(Follicle-stimulating hormone (FSH), Luteinizing hormone (LH))and Thyroid-stimulating hormone (TSH) are glycoprotein secreted by pituitary. Gonadotropin plays a key role in the regulation of the development and function of vertebrate gonads. TSH participates in development and metabolism by stimulate the thyroid to secrete T4 and T3. In the study, we prepared the prokaryotic expression plasmids encoding chicken TSHβ subunit, FSHβ subunit and LHβ subunit and produced the three recombinant proteins using RT-PCR and molecular approaches. We also purified the recombinant proteins by Metal Ion Affinity Chromatography. This work paves the way for future preparation of polyclonal antibodies against chicken TSHβ, FSHβ and LHβ in rabbits.