This study aimed to generate human papillomavirus (HPV) type 18 L1 virus-like particles (VLPs) and investigated the immunity effectiveness in vitro by using the Bac to Bac baculovirus expression system and Ni-NTA purification system to express and purify the VLP. The bio-function and immune response of purified VLP were analyzed by a mouse erythrocyte haemagglutination assay and the IFN-gamma ELISPOT. The proteins of interest, primarily located at the nucleus, formed VLP by self-assembly, and had an effective immune activity in vitro. The level of IFN-gamma in the specific HPV18 positive group was significantly higher than that of the mixed high-risk HPV infected group and non-HPV infection one in vitro. Our study indicated that HPV18 later protein L1 was efficiently expressed in Bac to Bac baculovirus expression system and the HPV18 L1-VLPs showed immunoreactivity and bio-activity in vitro. Furthermore, the ELISPOT results support the hypothesis that the cell-mediated immunity generated by HPV18 L1-VLP may cross-type among high-risk HPV types.