Despite the widespread and successful use of Newcastle disease (ND) vaccines, Newcastle disease virus (NDV) can seriously injure the reproductive tract of egg-laying hens, leading to rapid egg-drop and poor shell quality. Few published studies investigated local NDV-specific immune response in the reproductive tract after ND vaccination of hens. The present study investigated, for the first time, local NDV-specific antibody-mediated immunity in segments of the oviduct during the laying period. Specific pathogen-free (SPF) White Leghorn chickens were immunized following an ND vaccination programme applied in the field, which combined ND-attenuated vaccine (inoculated subcutaneously at one day, 2 weeks and 11 weeks of age) with inactivated vaccine (inoculated intramuscularly at 17 weeks). The infundibulum, magnum, isthmus and uterus (segments of the reproductive tract) were harvested at 28 weeks and 32 weeks of age (during the laying period). Supernatant from ex vivo tissue culture was collected and tested by: (i) haemagglutination inhibition (HI) test, (ii) commercial IDVet ND-enzyme-linked immunosorbent assay (ELISA) and (iii) NDV-specific IgG, IgM and IgA in-house ELISAs. For all sampling time points and oviduct segments, all samples were positive for commercial ND-ELISA and in-house ELISA-IgG. However, six of these ELISA-IgG positive samples yielded negative results when submitted to the HI test. Interestingly, NDV-specific IgM and IgA were detected frequently in the infundibulum and magnum as compared to the isthmus and uterus. These results show that the antibody immune response in the oviduct was induced by the timing of attenuated and inactivated ND vaccinations.