Objective To investigate the seroprevalence of tick-borne diseases in humans and domestic animals from rural areas of Zhejiang province. Methods Anji county, Jindong district and Tiantai county were selected for samples collection according to their geographic locations and historical prevalence of tick-borne diseases. Blood samples of humans and domestic animals were collected in the three sites. An indirect immuno-fluorescent antibody test was used to determine the presence of IgG antibodies of Rickettsiae heilongjiangii, Orientia tsutsugamushi, R. typhi, Anaplasma phagocytos, Ehrlichia chaffeensis, Bartonella, R. hainan and Coxiella burnetii in these samples.Results Six hundred and eighty-three blood samples including 579 from humans and 104 from domestic animals(53 from cattles and 51 from sheep)were collected from the three sites. Antibody positive rates of Orientia tsutsugamushi, R. typhi, Ehrlichia chaffeensis and Coxiella burnetii were significantly different between these sites. IgG from all the 8 pathogens were detected in samples from humans. It was found that the sero-prevalence rates of R. typhi, Bartonella and C. burnetii(20.7%,10.9%, 5.5%)of adults were higher than those of other Rickettsiae under investigation. The seroprevalence of R. typhi increased along with age. IgG from the 7 pathogens were detected in samples from domestic animals except for Anaplasma phagocytos. The sero-prevalence rates of R. typhi, Bartonella and R. hainan(69.2%, 51.0%, 22.1%)of adults were higher than those of other Rickettsiae investigated. Conclusion Tick-borne diseases did spread widely in humans and domestic animals from different rural areas of Zhejiang province. The sero-prevalence rates of R. typhi,B. henselae, R. hainan and C. burnetii were higher than that from other pathogens.