Neospora caninum, an apicomplexan protozoan causes economical losses due to reproductive failure associated with abortion among cattle. The diagnosis of neosporosis is difficult, due to nonspecific clinical signs and there are no parasitological examinations which can help to recognise neosporosis in vivo. Milk of seropositive cows was examined for both, IgG level to antigens of N. caninum and the presence of DNA of the parasite. Our results revealed that a milk sample from a seropositive cow diluted from 1:2 to 1:8 showed more than five times higher IgG level against N. caninum antigen when compared with a seronegative sample. Additionally, PCR test with Np6 and Np21 primers gave a 328 bp product in examined samples and confirmed the presence of N. caninum DNA in milk of seropositive cows. Data confirmed that vertical transmission of N. caninum from dams to older animals, in the late or post lactation period by milk should be taken into account. Moreover, the milk could be suitable in detection of N. caninum infection in cows using two complementary methods: ELISA and PCR.