Background: Prevention of mother-to-child transmission of HIV (PMTCT) is a frequently used word for programs and intervention methods to decrease the risk of mother-to-child transmission of HIV. The aim of this study was to identify determinants of the reduction of CD4 count through time and the maternal transmission of HIV to their child on the PMTCT program at health centers in North Shewa Zone, Ethiopia. Methods: The cohort study design was conducted by using secondary data collected from the cohort register of PMTCT starting from September 1, 2014 to November 30, 2017. In this study, a longitudinal study was conducted for two types of result; these were longitudinal response measurements of HIV infected women CD4 count and the time to maternal transmission of HIV taken from 203 patients. Results: The prevalence rate of HIV infection among exposed infants was 5.58%. Baseline CD4 count, visiting times, weight, and interaction between visiting time and baseline CD4 count had a statistically significant effect on the longitudinal biomarker. From the Weibull AFT model, ART start, partner test, clinical stage, educational status, place of delivery, and MUAC were statistically significant. Hence, as a measurement unit decreased in square root CD4 cell count by 1.18 elevates the risk of maternal transmission of HIV. Conclusion: In this study, the determinant of mother-to-child transmission of HIV including loss of weight, ART start (ANC), place of delivery at home, illiterate and mother with severe malnutrition, had a significant effect. The longitudinal biomarker also had a strong association with baseline CD4 and the risk of maternal transmission of HIV. Health education should be given about balanced diet, weight control, and take medication for HIV positive patients by the responsible bodies.