Seroepidemiology studies had been used to research the newly discovered human bocaviruses (HBoVs). Antibodies against the HBoV1-4 VP2 protein virus-like particles (VLPs) were found to be cross-reactive. The aim of the present study was to characterize the seroprevalence of HBoV1 and 2 among healthy populations in China. Recombinant HBoV1 and 2 VLPs were used to establish enzyme-linked immunosorbent assays (ELISAs) for detection of cross-reactivity between HBoV1 and HBoV2 in 1391 serum samples collected from healthy individuals in China. Of these, 884 samples were collected from Beijing and 507 were from Nanjing. Infection with HBoV1 and 2 was prevalent in healthy Chinese people, with the seroprevalence of HBoV1 and 2 in Beijing at 69.2 (612/884) and 64.4% (569/884), respectively. Highest seroprevalence was observed in 3-5-year-olds. The seroprevalence of HBoV1 was significantly decreased between 10-13-year-olds (80.3%) and 14-20-year-olds (62.3%, p< 0.05). For individuals over 20 years, seroprevalence was relatively constant at about 60%. Similar trends were observed in children from Nanjing, with seroprevalence of HBoV1 and 2 for healthy children at 80.7% (409/507) and 81.3% (412/507), respectively. Moreover, both mouse and human antibodies against HBoV1 and HBoV2 VLPs were found to be cross-reactive and 58.4% (813/1391) serum samples were seropositive for both HBoV1 and HBoV2. This finding suggests HBoV is highly prevalent in China and the antibodies produced as a result of infection with either HBoV1 or HBoV 2 will offer future protection. The cross-reactivity between HBoVs is crucial for accurately determining HBoV seroepidemiology.