The invention relates to a separation and purification method of recombinant hepatitis B core antigen, which comprises heat denaturation and clarification, ammonium sulfate precipitation, ultrafiltration and concentration, diafiltration and liquid replacement, depolymerization, the first step of molecular sieve chromatography, ultrafiltration and concentration, diafiltration And the steps of changing liquid, repolymerization and second step molecular sieve chromatography. The method can obtain recombinant hepatitis B core antigen with high purity, low host residue, uniform particle structure, and high stability, that is, the method has the characteristics of high antigen purity, low host protein and host nucleic acid residue, uniform antigen particles, and easy industrial scale-up.