The present work was designed to investigate bovine rotavirus antigen in fecal samples of forty diarrheic new borne calves of cattle (32) and buffaloes (8) below three months in some farms in Egypt. The detection was done by ELISA and indirect fluorescent antibody technique (FAT) as well as molecular method as real-time quantitative reverse transcriptase PCR (RT-qPCR) from samples collected during neonatal diarrhea. Six out of forty tested samples were found positive for rotavirus (15%) infection using ELISA test. Successful isolation of the rotavirus on MDBK cell line from two samples from the positive ELISA samples. The beginning of characteristic cytopathic effects (CPE) were observed from the 3rd passages as clumping, rounding of infected cells with syncytia and leaving empty vacuole space in MDBK cell line. The detected rotavirus with tissue culturing was identified by FAT with apple green fluorescence in the cytoplasm of infected cells. Viral RNA was extracted and the virus was confirmed by RT-qPCR assay utilized primers and probe that were designed to the target gene for rotavirus. The presence of rotavirus in fecal samples obtained from neonatal calves suggested its etiological roles and it is the main causative virus involved in neonatal calf diarrhea.