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Introduction: Approximately 6% of the deaths among children aged below five years in Tanzania is attributed to HIV/AIDS. Most of these deaths could be prevented if these children were identified, diagnosed and initiated on HIV treatment early. Despite the risk of acquiring HIV infection during the breastfeeding period, the uptake of Early Infant Diagnosis (EID) among HIV Exposed Infants (HEI), six weeks after cessation of breast feeding has received less attention in Tanzania. We assessed factors associated with uptake of EID among HIV Exposed Infants (HEI) in Tanzania. Methods: A facility based cross-sectional study was conducted in Iringa region from December 2015 to February 2016. HIV exposed children aged between 13.5 and 18 months were recruited for the study. Mothers and caregivers were interviewed using pretested structured questionnaires. Logistic regression analysis was used to assess factors influencing uptake of the test while controlling for potential confounders. Results: We recruited 387 HIV exposed children. Only 34.6% (95%, CI: 29.9%-39.6%) had received the HIV test. Having adequate knowledge on PMTCT [(aOR) = 5.1 (95% CI (2.26-11.64)], living in urban areas [(aOR=4.1 (95%, CI: 1.57-10.53)], awareness of the test before pregnancy [(aOR=4.5 (95% CI: 1.50-13.69)] or during pregnancy [(aOR=4.3 (95%, CI: 1.63-12.26)], and being attached to psychosocial support groups [(aOR=2.6 (95%, CI: 1.06-6.20)] were significantly associated with uptake of the test. Health-system related factors, unavailability of the test kits, and client related factor, long distance from home to facility and transport costs were common reasons for non-uptake. Conclusion: Uptake of the early infant diagnosis after breastfeeding cessation was found to be low. Socio-demographic factors including knowledge, awareness and place of residence were mostly associated with high uptake rate while low health system readiness was among the hindering factors. Tanzania PMTCT program needs to scale up early infant diagnosis services in rural areas, strengthen formation of psychosocial supportive groups and ensure availability of test kits in health facilities.