This study aims to prepare Nano-Graphene oxide (N-GO) for use as a new adjuvant to stimulate immune system against alpha-fetoprotein (AFP) antigen to generate AFP polyclonal antibody (Anti-AFP) to use for measuring AFP in human serum by radioimmunoassay system. The prepared N-GO as a new adjuvant compared with Freund's adjuvant. The study include two groups of New-Zealand rabbits. Two groups of New-Zealand rabbits are included in this study. Group I: immunized with AFP antigen emulsify with N-GO formula and group II: immunized with AFP antigen emulsify with Freund's adjuvant. The produced anti-AFP polyclonal were characterized in terms of titer, immunoresponse and displacement. The data of displacement % of group I immunized with AFP-N-GO formula were (79.5%, 75.4%, and 68.7%) and the data of displacement % of group II immunized with AFP- Freund's adjuvant were (68.7%, 62.8% and 58.7%) at dilution 1/16,000. 125I-AFP tracer carried out using Chloramine-T oxidation method. The prepared 125I-AFP tracer was separated and purified by PD-10 column. The assay's optimization was investigated. The standard curves for produced anti-AFP polyclonal for both of N-GO and Freund's adjuvant were studied, r = 0.996. In conclusion, N-GO proved to be as a new adjuvant formula is a more appropriate to stimulate immune system and produce polyclonal antibodies to use it as a basic component in assessment liquid phase radioimmunoassay to measured hormones or tumor marker such as AFP.