The β-subunit of human chorionic gonadotropin (hCGβ) is secreted by trophoblastic cells during normal pregnancy. In addition, hCGβ is also synthesized by many tumor and cancer cells, and it has been one of target molecules for active immunotherapy to prevent and treat hCGβ dependent tumors and cancers. The full coding region of hCGβ was isolated by the RT-PCR method, and was inserted into PCR3.1 eukaryotic expression vector to construct the recombinant PCR3.1-hCGβ DNA vaccine successfully. Using HeLa cells transient expression system, the capability of hCGβ expression of PCR3.1-hCGβ was confirmed in vitro, and the expressed hCGβ protein was mainly with intracellular state. With 20 μg PCR3.1-hCGβ plasmid DNA through intramuscular inoculation after bupivacaine-HCl inducing, the immunized BALB/c mice could express hCGβ antigen to induce both intensive hCGβ-specific humoral immune responses and CTL responses, and the antibody titer reached high to 1∶8000. Moreover, both two types of immune responses elicited by PCR3.1-hCGβ could attack HeLa cells to induce apoptosis in vitro. The results indicated that the immune responses induced by PCR3.1-hCGβ DNA vaccine have antitumor effects in vitro, and would be helpful to detect the antitumor effects of PCR3.1-hCGβ DNA vaccine in vivo.